Isoalantolactone mediates the degradation of BCR-ABL protein in imatinib-resistant CML cells by down-regulating survivin

ABSTRACT

Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone’s anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5^T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5^T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML.

KEYWORDS:

• Isoalantolactone
• survivin
• BCR-ABL
• chronic myeloid leukemia
• protein degradation
• caspase-3

1. Introduction

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that results in the formation of the BCR-ABL fusion gene, which encodes the constitutively activated BCR-ABL tyrosine kinase [1–3]. The current treatment for CML (CML) with tyrosine kinase inhibitors (TKI) is effective but not curative in many patients, and it is frequently limited by drug resistance [4,5]. Survivin expression has been linked to increased tumor resistance to a variety of chemotherapy agents. Patients with undetectable survivin levels had cytogenetic complete remissions, whereas patients clinically resistant to imatinib had elevated survivin levels, similar to those seen in imatinib-resistant cell lines [6]. The survivin protein levels were significantly reduced in an imatinib-sensitive CML cell line at the same time [7–9]. Survivin plays an important role in the maintenance of CML drug resistance, but no natural compounds targeting survivin protein have been found to overcome TKI resistance in CML.

Natural compounds have medicinal potential and are a valuable source for the development of targeted regulation of BCR-ABL proteins. Diosmetin, a phytoestrogen, inhibits the phosphorylation of BCR-ABL and has a minor effect on BCR-ABL levels [10]. By blocking the BCR-ABL signaling pathways, CD-200 induces apoptosis and has an anti-proliferative effect in BCR-ABL ^T315I cells, which are resistant to imatinib [11]. Tambjamines are natural compounds isolated from marine invertebrates with a variety of pharmacological activities [12–14]. T21, a novel indole-based tambjamine analog, has recently been identified as a promising antitumor agent that modulates the expression of apoptotic proteins, such as survivin [15]. To that end, searching for candidate compounds targeting survivin downregulation among natural compounds may yield lead compounds to overcome TKI resistance. Isoalantolactone, a major sesquiterpene lactone isolated from the root of Inula helenium L. It is most commonly used in the treatment of chronic gastritis, gastrointestinal dysfunction, intercostal neuralgia, and chronic inflammation [16–19]. In vitro studies have revealed that the drug has a potent anti-tumor activity in lung carcinoma, pancreatic carcinoma, prostate adenocarcinoma, and breast carcinoma by inducing apoptosis, autophagic cell death, or inhibiting migration and invasion [20–24]. Isoalantolactone has been shown to induce apoptosis in human leukemia drug-resistant cell-line K562/A02 cells overexpressing the BCR-ABL oncogene by downregulating BCR-ABL and Bcl-2, as well as induced activation of Bax, cytochrome c, caspase-9, caspase-3, and PARP [25]. It remains to be seen whether isoalantolactone can overcome TKI resistance by targeting the survivin protein.

To investigate the effect of isoalantolactone, we treated imatinib-sensitive and imatinib-resistant cells KBM5 and KBM5^T315I with different concentrations of isoalantolactone and examined cell proliferation and apoptosis. We discovered that isoalantolactone-induced BCR-ABL degradation in a caspase-3 dependent manner via targeting survivin degradation. These findings suggest that isoalantolactone is a candidate natural compound that could be used to treat TKI-resistant CML.

2. Materials and methods

2.1. Chemicals

A stock concentration of 20 mM isoalantolactone was provided by Dr. Wu Ying-Li (Department of Pathophysiology, Shanghai Jiaotong University School of Medicine, Shanghai, China). It was isolated from the root of Inula helenium L. The batch number is Y-059-131027. HPLC is used as a test criterion. The pan-caspase inhibitor Z-VAD-FMK (catalog no. S7023; Selleck Chemicals), MG-132 (catalog no. s2619; Selleck Chemicals), CQ (catalog no. C6628; Sigma), 3 MA (catalog no. M9281; Sigma) was dissolved in DMSO and kept at −80°C.

2.2. Cell culture.

Dr. Wu Ying-Li provided K562, K562R, KBM5, and KBM5^T315I cells. At 37°C and 5% CO₂, KBM5 cells were cultured in RPMI-1640 (catalog no. SH30809.01B; HyClone) with 10% FBS (catalog no. 10091148, Gibco; Thermo Fisher Scientific, Inc), 100 U penicillin, and 100 g/L streptomycin (catalog no. 15070063, Gibco; Thermo Fisher Scientific, Inc). KBM5^T315I cells were cultured in 10 μM imatinib medium.

2.3. Cell proliferation assay

2000 cells in 100 μl were incubated in each well to assess cell viability and determine the study’s IC50 dose. The treatment group included 2.5, 5, 10, 20, 40, and 80 μmol/L isoalantolactone, while the control group included dimethyl sulfoxide (DMSO). After 12, 24, 36 and 48 h, 10 μl of CCK8 solution was added to each well of the plate, and the wells were incubated for 2 h. Each well’s absorbance at 450 nm was used to calculate the cell viability. The following equation calculated cell viability (%): = 100 × (mean OD of test wells/mean OD of control wells).

2.4. Flow cytometry

The apoptosis was determined by Annexin V-FITC/PI, Annexin V-Alexa Fluor 647/PI staining (we chose 647/PI staining for apoptosis detection because we transfected shRNA with a GFP tag). Briefly, the cells were treated with isoalantolactone for 24 h. Then, cells were centrifuged at 1,000 rpm for 5 min, and washed with a binding buffer. Following this, cells were incubated in working solution (100 μl binding buffer with 5 μl Annexin V-FITC/Alexa 647 and 5 μl PI) for 15 min in the dark.

2.5. Western blotting.

Cell pellets were lysed with 150 μl lysis buffer [50 mM Tris-HCl (pH6.8), 100 mM DTT, 2% SDS, 10% glycerol and ddH2O]. The cell lysates were denatured at 100°C for 10 min. The protein concentration of the cell lysate was measured using a spectrophotometer. The total protein (100 μg) of the cell lysate was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with 5% skim milk (catalog no. A600669, Sangon Biotech, China) for 1 h at room temperature. The membrane was incubated overnight at 4°C with the following appropriate primary antibodies: Anti-cleaved caspase-3 (catalog no. 9664, Cell Signaling Technology, Inc.), anti-caspase-3 (catalog no. ab184787, Abcam), anti-caspase-7 (catalog no. 9492, Cell Signaling Technology, Inc.), anti-c-Abl (catalog no. 2862, Cell Signaling Technology, Inc.), anti-cleaved PARP (catalog no. 5625, Cell Signaling Technology, Inc.), anti-survivin (catalog no. ab76424, Abcam) anti-Ub (catalog no.3932, Cell Signaling Technology, Inc) anti-Flag (catalog no.14793S, Cell Signaling Technology, Inc) anti-β-actin (catalog no. 4970, Cell Signaling Technology, Inc.). The membranes were then incubated with HRP-conjugated rabbit anti-mouse antibody (catalog no. 315-035-003, Jackson Immuno Research Laboratories, Inc.) for 1 h. ECL reagent (catalog no. WBKLS0500, EMD Millipore) was used to detect the signals.

2.6. RNA extraction and reverse transcription-quantitative PCR (RT-Qpcr)

Total RNA was extracted from cells using RNAiso Plus reagent (catalog no. 9109, Takara, Bio, Inc.) according to the manufacturer’s protocol. RNA (500 ng) was reverse transcribed to cDNA using the PrimeScript RT reagent kit (catalog no. RR047A, Takara, Bio, Inc.). qPCR analysis was conducted on a Roche Light Cycler480 machine with SYBR Green (catalog no. 11202ES03, Yeason). Relative expression was determined using the 2^−ΔΔCq method with ACTB as the reference. The primers were synthesized as follows: BCR-ABL forward, 5´- CGG GAG CAG CAG AAG AAG TGT-3´ and reverse, 5´- CGA AAA GGT TGG GGT CAT TTT C-3´; Survivin forward, 5´-TGA ACT TCA GGT GGA TGA GGA GA −3´ and reverse, 5´-GTC TAA TCA CAC AGC AGT GGC AA −3´; and ACTB forward, 5´-CAT GTA CGT TGC TAT CCA GGC-3´, and reverse, 5´-CTC CTT AAT GTC ACG CAC GAT-3´. All experiments were performed three times independently.

2.7. Transfection and viral infection

Dr. YingLi Wu provided the pSIN4-EF1alpha-IRES plasmids (Vector backbone identical to #61062, addgene). Survivin’s coding sequence was amplified by PCR using Jurkat CDNA as the template and the PrimeSTAR GXL DNA Polymerase Kit (#R050Q, TaKaRa, Japan). The following primers were used in the PCR: F: 5´ -GGG CTA GCT AGC TAG GAA TTC ATG GAC TAC AAA GAC GAT GAC GAC AAG GAC TAC AAA GAC GAT GAC GAC AAG GAC TAC AAA GAC GAT GAC GAC AAG ATG GGT GCC CCG ACG TTG-3´. R: 5´-GCC CTA GAT GCA TGC GGA TCC TCA ATC CAT GGC AGC CAG CTG-3´. 0.5 μg CDNA, 2 μl forward primers (10 μM), 2 μl reverse primers (10 μM), 4 μl dNTP Mixture, 10 μl 5 ×PrimeSTAR GXL Buffer (Mg²⁺ plus), 2 μl PrimeSTAR GXL DNA Polymerase (1.25 U μl), and RNase Free ddH₂O were used in the PCR. Pre-denaturation at 98°C for 60 s, denaturation at 98°C for 10 s (30 cycles), annealing at 55°C for 15 s, and the extension at 68°C for 90 s were the reaction conditions. The PCR products were separated on a 1% agarose gel and purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (#9762, TaKaRa, Japan). The restriction enzymes BamH I (#1605, TaKaRa, Japan) and EcoR I (#1611, TaKaRa, Japan) were used to digest the pSIN plasmids. The reaction was carried out in a 50 μl volume, with 1 μg pSIN plasmids, 1 μl BamH I, 1 μl EcoR I, 5 μl 10×QuickCut Buffer, and RNase Free ddH₂O. The reaction conditions were as follows: 30 min of digestion at 37°C. After stopping the reaction with 0.8% agarose gel electrophoresis, the linearized pSIN plasmids were detected and purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0. To obtain recombinant plasmids coding survivin, the ligation reaction of PCR products and linearized pSIN plasmids was catalyzed by the ClonExpress II One-Step Cloning Kit (#C11201, Vazyme, China). The reaction was carried out in a 20 μl volume with 160 ng linearized pSIN plasmids, 60 ng PCR products, 2 μl Exnase II, 4 μl 5×CE II Buffer, and RNase Free ddH₂O. The reaction conditions were as follows: ligation at 37°C for 30 min, followed by 5 min on ice. The recombinant plasmids were amplified and DNA sequenced to confirm their existence (GENEWIZ, China) The Lipofectamine 3000 Transfection Kit (#L3000015, Invitrogen, USA) was used to transfect cells (50–60% cell confluent monolayer in one well of a 6 well plate) with pSIN or pSIN-Flag3-Survivin plasmids. The steps were as follows: Mix I is made up of 6 μl Lipofectamine 3000 Reagent and 250 μl OptiMEM Medium (#31985062, gibco). 2 μg pSIN/pSIN-Flag3-Survivin plasmids, 4 μl P3000 Reagent, and 250 μl OptiMEM Medium make up the Mix II solution. The above solutions I and II were thoroughly mixed and allowed to stand at room temperature for 5 min. The mixture was then added to one well of a 6-well plate containing 1.5 ml of normal medium. Transfected cells can then be used in subsequent experiments. All experiments were carried out three times independently and yielded similar results.

ShRNA sequences targeting survivin, caspase 3, caspase 7, and a negative control sequence were synthesized, annealed, and inserted into lentiviral pLL3.7 vectors. The following were the target sequences: survivin-1, 5´- GGC TGT TCC TGA GAA ATA A −3´; Survivin-2, 5´- GGA GAC AGA ATA GAG TGA T −3´; caspase-7–1, 5´-GGC AAA TGC ATC ATA ATA A-3´; caspase- 7–2, 5´-GCC CAT CAA TGA CAC AGAT-3´; caspase-3–1,5´-CCC TGG ACA ACA GTT ATA A-3´; caspase-3–2,5´-GCG TGA TGT TTC TAA AGA A-3´; and scrambled, 5´-GTT CTC CGA ACG TGT CAC GT-3´. 293T cells were transfected in Opti-MEM® I (Gibco; Thermo Fisher Scientific, Inc.) using Lipofectamine® 3000 (Thermo Fisher Scientific) once they were 70% confluent, according to the manufacturer’s protocol. The medium was changed to complete DMEM-H 8 h after transfection. Viral medium was created by co-transfecting 293T cells with shRNA expressing pLL3.7 lentiviral vectors as well as psPAX.2 and pMD2. G (Addgene, Inc.). In a 2:1:1 ratio, vectors were transfected. The viral particles were harvested 48 h after transfection, filtered through a 0.22 μm filter, and used for infection. KBM5 and KBM5-T315I cells were incubated with lentivirus-containing medium and Polybrene (10 g/ml). After 24 h, the culture medium was replaced with a complete medium, and cells expressing GFP were chosen.

2.8. Immunoprecipitation

Cells (70–80% cell confluent monolayer in a 6 cm cell culture dish) were washed twice with PBS buffer before being added to 1 ml RIPA lysis buffer (#P0013D, Beyotime Biotech, China) containing protease and phosphatase inhibitors (#B14001, Bimake). The cell lysate was then placed on ice for 60 min while being vibrated every 2 min. The cell lysate was then treated with an ultrasonic cell disruptor (power: 200 W; mode: work 4 s, stop 3 s, 5 times) (#DH92IIN, LAWSON, China). The supernatant of the cell lysate was collected and divided into two parts: one (350 μL) for the target protein immunoprecipitation assay and the other (200 μl) for total protein analysis. The immunoprecipitation assay protocol was as follows: Gently invert the resin to thoroughly resuspend it. Check that the ANTI-FLAG M2 Magnetic Beads (#M8823, Sigma) are in a uniform suspension in the bottle. 10 μl beads should be removed for use. Re-suspend the beads in 200 μl TBS to re-equilibrate them (50 mM Tris HCl, 150 mM NaCl, pH 7.4). To collect the beads, place the tube in an appropriate magnetic separator. Remove the storage buffer/TBS mixture and discard it. At 4°C, incubate the protein extract with the equilibrated beads for 34 h. Once the binding is finished, collect the magnetic beads by placing the tube in the appropriate magnetic separator and removing the supernatant. To remove all nonspecifically bound proteins, wash the resin beads with TBS buffer. The beads were then washed with TBS, and the protein binding to the beads was eluted with 30 μl SDS lysis buffer. Following that, the Western Blot protocol was carried out. All experiments were carried out three times independently and yielded similar results.

2.9. Cellular Thermal Shift Assay (CETSA)

×10 [7] cells were cultured in 10 cm dishes for 24 h to produce a CETSA in HEK293 cell lysates. The cells were washed in PBS before being harvested in 1 ml of PBS [1% proteinase inhibitor cocktail]. After 3 min of incubation in Liquid Nitrogen and melting at room temperature, the process was repeated three times. The lysates were centrifuged for 30 min at 14,000 g at 4°C, and the supernatants were separated into two tubes. (1) The melt curve “CETSA” approach: The supernatants and 100 μM Isoalantolactone were incubated at room temperature for 30 min. Control cells were exposed to an equal volume of DMSO as a vehicle. Proteins from the two tubes were then divided equally into six 100 μl tubes and placed in the PCR instrument, which was set to the indicated temperatures. (2) The isothermal dose–response (ITDR) CETSA: The supernatants and Isoalantolactone were incubated with the indicated concentration (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM), and placed in the PCR instrument, 50°C 3 min, 25°C 3 min. The lysates were centrifuged at 14,000 g for 30 min at 4°C, and the supernatants were transferred to new tubes and incubated for 10 min at 100°C with an equal volume Lysis buffer. The supernatants were analyzed by Western blot.

2.10. Clinical samples collection

We collected bone marrow cells from four CML patients and treated them for 48 h with 20 μM isoalantolactone. All procedures were carried out in accordance with the ethical guidelines established by the Ethics Committee of Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, and written informed consent was obtained.

2.11. Statistical analysis

The statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.) or SPSS 21.0 (IBM Corp.). Compared isoalantolactonens with the groups for cell viability and mRNA relative expression were assessed by one-way AVONA and Dunnett’s multiple comparison test. The apoptosis rates and protein expression were assessed by students’ t-test. P < 0.05 was considered to indicate a statistically significant difference.

3. Results

3.1. Isoalantolactone inhibits the proliferation and induces apoptosis of imatinib- resistant CML cells

Imatinib-sensitive cell lines (KBM5 and K562) and imatinib-resistant cell lines (KBM5^T315I and K562R) were used for testing, as shown in Figures (Figure 1b and Supplementary Figure S1A). The proliferation of KBM5, K562, KBM5^T315I, and K562R cells was inhibited by isoalantolactone in a dose and time-dependent manner, with IC50 values of 8.58, 5.04, 15.13, and 19.32 μM at 48 h (Figure 1(c,dD) and Supplementary Figure S1B, C). When 20 μM isoalantolactone was added to KBM5, K562, KBM5^T315I, and K562R cells, the amount of Annexin V+/PI+ (late apoptosis), and Annexin V-/PI+ (necrosis) stained cells were increased (Figure 1(e,f) and Supplementary Figure S1D, E). This suggests isoalantoactone-induced apoptosis in both imatinib-sensitive and imatinib-resistant cells.

Figure 1. Isoalantolactone inhibits the proliferation and induces apoptosis of imatinib-sensitive and resistant CML cells.

Note: (a) Isoalantolactone’s chemical structure (b) KBM5 and KBM5^T315I cells were treated with imatinib at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5 μM) for 24 h. The cell viability was determined by CCK8 method. (c and d) KBM5 cells and KBM5^T315I cells were treated with different concentrations of isoalantolactone at different concentrations (0, 2.5, 5, 10, 20, 40, 80 μM) for 0, 12, 24, 36, 48 h, the cell viability was measured by the CCK8. (e and f) After KBM5 cells and KBM5^T315I cells were treated with or without 20 μM isoalantolactone for 24 h and cell apoptosis was detected by Annexin V-FITC/PI double staining with flow cytometry. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01.

3.2. Isoalantolactone downregulates BCR-ABL and survivin protein levels, but it can’t alter the mRNA levels of survivin and BCR-ABL in imatinib-resistant CML cells

We investigated whether isoalantolactone could affect survivin and BCR-ABL protein levels in imatinib-sensitive or imatinib-resistant CML cells. We discovered that isoalantolactone significantly reduced survivin and BCR-ABL protein levels in a dose and time-dependent manner (Figure 2(a–d)and Supplementary Figure S1F, G). Isoalantolactone was applied to imatinib-sensitive cells (KBM5 and K562) and imatinib-resistant cells (KBM5^T315I and K562R) for 24 h at concentrations of 0, 10, 15, and 20 μM. The detection of cleaved-caspase-3 and cleaved-PARP indicated that isoalantolactone could induce apoptosis in imatinib-sensitive cells as well as imatinib-resistant cells (Figure 2(a–d) and Supplementary Figure S1F, G). At the same time, we used clinical samples to show that 20 μM isoalantolactone could significantly inhibit survivin protein in patients with both early-stage and drug-resistant CML (Supplementary Figure S2A-C). However, no significant changes were identified in survivin and BCR-ABL mRNA levels (Figure 2(e–h)), indicating that the down-regulation of survivin and BCR-ABL induced by isoalantolactone was not at the transcriptional level.

Figure 2. Effect of isoalantolactone on survivin and BCR-ABL protein and mRNA levels in imatinib-sensitive and resistant CML cells.

Note: (a and b) KBM5 or KBM5^T315I cells were treated with the indicated concentrations of isoalantolactone for 24 h, and survivin and BCR-ABL protein levels were detected by western blotting (see Supplementary Figure S3A and B). (c and d) KBM5 or KBM5^T315I cells were treated with isoalantolactone 20 μM for 0, 6, 12 and 24 h, and survivin and BCR-ABL protein levels were detected by western blotting (see Supplementary Figure S3C and D). (e, f, g and h) RT-PCR was used to detect the changes of BCR-ABL and survivin mRNA levels in KBM5 cells and KBM5^T315I cells treated with isoalantolactone for 24 h. ns, no significance.

3.3. The survivin mediates the apoptosis and down-regulation of BCR-ABL protein in imatinib-resistant CML cells

In imatinib resistant and sensitive CML cell lines, we established survivin knockdown cell lines and found that the BCR-ABL protein level of survivin knockdown cells was significantly reduced, which confirmed that the down-regulation of BCR-ABL was mediated by the reduction of survivin (Figure 3(a,b)). At the same time, apoptosis was significantly increased in CML cells that knocked down survivin (Figure 3(c–d)). These findings suggest that survivin is responsible for apoptosis and BCR-ABL protein downregulation in KBM5 and KBM5^T315I cells.

Figure 3. The degradation of BCR-ABL protein in imatinib sensitive and resistant CML cells through down-regulation of survivin.

Note: (a and b). KBM5 cells and KBM5^T315I cells were transfected with survivin shRNA sequence to construct survivin knockdown cells and BCR-ABL and survivin protein levels were detected by western blotting (see Supplementary Figure S4A and B). (c and d). KBM5 cells and KBM5^T315I cells were transfected with survivin shRNA sequence and cell apoptosis was detected by Annexin V-Alexa Fluor647/PI or Annexin V-FITC/PI double staining with flow cytometry. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01.

3.4. Isoalantolactone mediates imatinib resistance and sensitive CML cells apoptosis through down-regulation of survivin

We constructed survivin overexpressed cell lines to investigate if isoalantolactone mediates imatinib resistance and sensitive cell-line apoptosis by down-regulating survivin. The overexpressed survivin in imatinib sensitive and resistant cells could resist the down-regulation of BCR-ABL protein induced by isoalantolactone (Figure 4(a,b)). Treating survivin overexpressed KBM5 and KBM5^T315I cells with 20 μM isoalantolactone, the apoptosis was significantly inhibited (Figure 4(c,d)).

Figure 4. Isoalantolactone mediated imatinib resistance and sensitive CML cells apoptosis through down-regulation of survivin.

Note: (a and b) PSIN or pSIN-Flag3-survivin plasmids were transfected into KBM5 cells and KBM5^T315I cells that were treated with 20 μM isoalantolactone or not. Survivin and BCR-ABL protein levels were detected by western blotting (see Supplementary Figure S5A and B). (c and d) The overexpressed cells and cell apoptosis were detected by Annexin V-Alexa Fluor647/PI or Annexin V-FITC/PI double staining with flow cytometry. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01.

3.5. Isoalantolactone enhances ubiquitin-dependent degradation of survivin through direct interaction with surviving protein.

In order to understand the mechanism of survivin protein degradation in CML cells induced by isoalantolactone, both KBM5 and KBM5^T315I cells were pretreated with the autophagy inhibitor 3-MA and the lysosomal inhibitor CQ in the presence or absence of isoalantolactone, which revealed no attenuation of survivin protein degradation, indicating that the autophagy pathway was not responsible for the isoalantolactone-induced survivin protein degradation (Figure 5(a–d)). Following that, KBM5 and KBM5^T315I cells were treated with the proteasome inhibitor MG132 before being incubated with isoalantolactone. After treatment with MG132, survivin protein was restored, which means that the proteasome pathway may participate in the degradation of survivin Figure 5(e,f). The immunoprecipitation test suggested that isoalantolactone may cause survivin degradation by increasing survivin ubiquitination (Figure 6a). However, it is unknown whether isoalantolactone directly targets survivin. We used CETSA to perform target validation to see if isoalantolactone directly binds to survivin. This method is based on the idea that ligand binding increases the thermal stability of a target protein. We discovered that when HEK293T cell lysates with overexpressed survivin were incubated with isoalantolactone, the thermal stability of survivin was reduced when compared to the control group, indicating that isoalantolactone may trigger survivin thermal stabilization via direct binding Figure 6(b,c).

Figure 5. The proteasome pathway involved in isoalantolactone-induced down-regulation of survivin protein in imatinib-sensitive and resistant CML cells.

Note: (a and b) Autophagy inhibitor 3-MA was used alone or in combination with isoalantolactone to treat KBM5 and KBM5^T315I cells, the protein level of survivin was detected by western blotting (see Supplementary Figure S6A and B). (c and d) The autophagy inhibitor CQ was used alone or in combination with isoalantolactone to treat KBM5 and KBM5^T315I cells. Western blotting was used to detect the protein levels of survivin protein (see Supplementary Figure S6C and D). (e and f) KBM5 and KBM5^T315I cells were treated with the proteasome inhibitor MG132 alone or in combination with isoalantolactone. Western blotting detected the protein level of survivin in KBM5 and KBM5^T315I cells (see Supplementary Figure S6E and F).

Figure 6. Isoalantolactone enhanced survivin protein ubiquitin modification by directly binding to survivin protein.

Note: (a) pSIN-Flag3-Survivin plasmids were transfected into HEK293T cells, and 48 h after transfection cells were treated with 20 μM MG132 for 2 h and with 0, 10, 15 μM isoalantolactone for 6 h, respectively. Then, Flag was immunoprecipitated from the cell lysate, and the ubiquitination of Flag was assessed by western blotting analysis (see Supplementary Figure S7A). (b) pSIN-Flag3-Survivin plasmids were transfected into HEK293T cells. CETSA was used to determine isoalantolactone in HEK293 cell lysates. The results of immunoblotting showed the thermostability of survivin following heat treatment at the indicated temperatures in the presence (+) or absence (−) of isoalantolactone (100 μM) (see Supplementary Figure S7B). (c) pSIN-Flag3-Survivin plasmids were transfected into HEK293 cells. They were incubated with the indicated concentrations of isoalantolactone. Dose-dependent stabilization was observed in HEK293Tells (see Supplementary Figure S7C).

3.6. Isoalantolactone induced BCR-ABL protein degradation is caspase-3 dependent in imatinib sensitive and resistant cells

KBM5 and KBM5^T315I cells were treated with caspase inhibitor Z-VAD-FMK alone or in combination with isoalantolactone to determine the mechanism of isoalantolactone-induced BCR-ABL degradation in CML cells, which revealed that the inhibitor Z-VAD-FMK blocked the caspase-3 activation induced by isoalantolactone Figure 7(a,b). Furthermore, cotreatment with isoalantolactone and Z-VAD-FMK partially rescued BCR-ABL protein expression in KBM5 and KBM5^T315I cells Figure 7(a,b), indicating that isoalantolactone may downregulate BCR-ABL protein expression via caspase activation. However, this method had no effect on survivin protein expression. Caspase-3 and caspase-7 were knocked down in KBM5 and KBM5^T315I cells to confirm that caspase activation contributes to isoalantolactone-induced BCR-ABL protein degradation. Caspase-7 knockdown could not rescue isoalantolactone-induced BCR-ABL protein degradation Figure 7(c,e), whereas reducing caspase-3 could partially rescue isoalantolactone-induced BCR-ABL protein degradation Figure 7(d,f). These findings suggest that isoalantolactone-induced BCR-ABL protein degradation in imatinib-resistant CML cells was partially dependent on caspase-3 activation.

Figure 7. The down-regulation of BCR-ABL protein induced by isoalantolactone depends on the activation of caspase-3 in imatinib-resistant and sensitive CML cells.

Note: (a and b) KBM5 and KBM5^T315I cells were treated with caspase inhibitor Z-VAD or combined with isoalantolactone for 12 h, and the protein levels of caspase-3, BCR-ABL, and survivin were detected by western blotting (see Supplementary Figure S8A and B). (c and d) Caspase-7 shRNA-transfected, Caspase-3 shRNA-transfected and EV-transfected KBM5 cells were treated with isoalantolactone for 12 h. Western blotting was used to detect the changes of caspase-7, caspase-3, BCR-ABL protein levels (see Supplementary Figure S8C and D). (e and f) Caspase7 shRNA-transfected, Caspase-3 shRNA-transfected, and EV-transfectedKBM5^T315I cells were treated with isoalantolactone for 12 h. Western blotting was used to detect the changes of caspase-7, caspase-3, BCR-ABL, and survivin protein levels (see Supplementary Figure S8E and F).

4. Discussion

LY2181308 is a single-strand antisense oligonucleotide that targets survivin by binding to and degrading its mRNA, preventing it from being translated into protein. In several tumor cell lines and human tumor xenografts, LY2181308 significantly reduced survivin mRNA and protein, as well as cell-cycle arrest, cell-death induction, and tumor growth inhibition [26]. Another antisense oligonucleotide, SPC3042 (EZN-3042), was identified as a new agent with greater potency for survivin mRNA inhibition than previous antisense agents, including LY2181308 [27]. SPC3042-induced cell cycle arrest, pronounced cellular apoptosis, and sensitization of prostate cancer cells to taxol treatment in vitro and in vivo. Survivin has emerged as an ideal target for oncology drug discovery, but no potential small molecule survivin inhibitor has been found [28]. In this study, isoalantolactone inhibited proliferation and promoted apoptosis in both imatinib-sensitive cells (KBM5 and K562) and imatinib-resistant CML cell lines (KBM5^T315I and K562R) in a dose- and time-dependent manner). We discovered that isoalantolactone inhibited survivin, which mediated BCR-ABL protein degradation in a caspase-3 dependent manner.

Whether CML cells are sensitive to imatinib or not, inhibition of survivin expression will lead to cell growth arrest and subsequent cell death [6]. It suggests that suppressing survivin expression may have a therapeutic effect in patients with imatinib-resistant CML. We discovered that isoalantolactone induced the degradation of survivin and BCR-ABL, particularly in T315I mutated cells, but did not affect BCR-ABL and survivin at the mRNA level. The survivin protein changes can be reproducible in clinical samples. It was discovered that isoalantolactone mediated BCR-ABL degradation via survivin protein disruption, which may be the key reason for isoalantolactone’s ability to overcome BCR-ABL T315I cell resistance. On the other hand, more information about the interaction of isoalantolactone and survivin needs to be clarified.

The ubiquitin/proteasome pathway and the lysosomal pathway are two important intracellular protein degradation pathways [29,30]. However, in the current study, the proteasome inhibitor MG132, the autophagy inhibitor CQ, and the lysosomal inhibitor 3-MA were unable to reduce isoalantolactone-induced survivin and BCR-ABL protein degradation, indicating that these two pathways are not involved in isoalantolactone-induced survivin and BCR-ABL protein downregulation. The proteasome inhibitor MG132 can rescue isoalantolactone-induced survivin downregulation but not BCR-ABL protein, implying that the ubiquitin/proteasome pathway involved in survivin degradation.

Survivin overexpression in HEK293 cells treated with isoalantolactone resulted in increased survivin ubiquitination, implying that isoalantolactone can degrade survivin by increasing survivin ubiquitination. CETSA then observed isoalantolactone possibly binds and interacts with the surviving protein itself, presumably binding isoalantolactone. Survivin protein degradation may be aided by the intracellular ubiquitin proteasomal degradation pathway. This is analogous to M×106‘s direct interaction with survivin, which selectively degrades survivin via proteasome-mediated degradation [31]. This demonstrates that isoalantolactone is directly targeted as an inhibitor to induce enhanced degradation of survivin protein, though it remains unknown how it interacts with survivin protein and how it increases the level of ubiquitin modification of survivin protein.

Caspase activation has been shown in the literature to downregulate BCR-ABL protein [32,33]. In this study, we discovered that isoalantolactone activated caspase-3 and increased cleaved-caspase-3 accumulation. PARP cleavage was induced after caspase activation [34]. Furthermore, caspase inhibitors were found to reduce isoalantolactone-induced BCR-ABL degradation. These findings suggest that isoalantolactone can inhibit BCR-ABL protein expression by activating caspases. Caspase-3 and caspase-7 are thought to be involved in protein degradation, according to previous research [35]. For example, activated caspase-3 promotes FTase/GGTase-subunit cleavage, resulting in the accumulation of unpropylated G-proteins. WT1 protein can also be degraded by Caspase-3 [36]. Furthermore, caspase-7 has been shown to be capable of cleaving TNFRI [37]. Caspase-3 and caspase-7 are also involved in BCR-ABL degradation induced by p53 [4]. To investigate the molecular mechanism of isoalantolactone-induced BCR-ABL degradation, we knocked down caspase-3 expression in KBM5 and KBM5^T315I cells and discovered that caspase-3 knockdown can partially rescue isoalantolactone-induced BCR-ABL degradation. Caspase 7 inhibition did not restore BCR-ABL degradation. This could be due to the fact that caspase-3 and caspase-7 have different activities toward substrate proteins. Caspase-3 has been reported to be more promiscuous than caspase-7 and to be the primary executioner caspase during the demolition phase of apoptosis [35]. However, the detailed molecular mechanism of BCR-ABL fusion protein degradation by activated caspase-3 remains unknown.

TKI resistance during CML treatment is urgently needed in the search for new anti-resistance strategies. According to current reports, most studies are focused on the discovery of TKI that can either downregulate BCR-ABL transcription or inhibit tyrosine kinase activity. We propose that isoalantolactone inhibits survivin by the ubiquitin proteasome pathway and mediates BCR-ABL downregulation in a caspase-3 dependent manner (Figure 8). This provides a potential candidate compound as well as a hint for potential new strategies to overcome different types of TKI resistance.

Figure 8. The schematic diagram of the effect and mechanism of isoalantolactone in the process of overcoming imatinib resistance.

Note: Isoalantolactone induces the down-regulation of survivin and initiates apoptosis. In turn, the BCR-ABL protein is degraded by caspase-3 activation to overcome the imatinib resistance.

Authors’ contributions

Shan-Shan Yin: Conceptualization, Methodology, Writing – original draft preparation. Chen Chen: Conceptualization, Methodology, Writing – original draft preparation. Zhen Liu: Methodology. Shan-Ling Liu: Methodology, Resources. Chao Zhang: Supervision, Project administration. Quan-Wu Zhang: Supervision, Project administration. Feng-Hou Gao: Supervision, Project administration, Writing – review and editing, Conceptualization.

Ethics approval and consent to participate

All procedures were carried out in accordance with the ethical guidelines established by the Ethics Committee of Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, and written informed consent was obtained.

List of abbreviations

CML	=	Chronic myeloid leukemia
TKI	=	Tyrosine Kinase Inhibitors
IC50	=	half maximal inhibitory concentration
CESTA	=	Cellular Thermal Shift Assay

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2023.2209963.

Additional information

Funding

This work was supported in part by the National Natural Science Foundation of China (82002922), Science and Technology Committee of Shanghai (14401901500).