Alantolactone inhibits cell autophagy and promotes apoptosis through targeting AP2M1 in acute lymphotic leukemia

Background: Acute lymphoblastic leukemia (ALL) is an aggressive hematopoietic malignancy that is most common in children. Alantolactone (ALT) has been reported to have antitumor activity in different types of cancers. This study aimed to investigate the antitumor activity and molecular mechanism of ALT in ALL. Methods: The ALL cell lines were treated with 1, 5 and 10μM of ALT, and then subjected to MTT assay and RNA sequencing. Flow cytometry, JC-1 staining and immunouorescence staining assays were employed to measure cell apoptosis and autophagy. Meanwhile, Western blot analysis was used to detect apoptosis and autophagy related proteins. Finally, the effect of ALT on tumor growth was measured in BV173 xenograft nude mouse model. Results: In this study, we demonstrated that ALT inhibited the proliferation of ALL cells in does-dependent manner. A series of experiments demonstrated that ALT inhibited cell proliferation, colony formation, autophagy, induced apoptosis and restained tumor growth in vivo through upregulating adaptor related protein complex 2 subunit mu 1 (AP2M1). Moreover, autophagy activator rapamycin attenuated the pro-apoptotic effect of ALT on BV173 and NALM6 cell lines. Further, overexpressed AP2M1 decreased the expression of Beclin1, LC3-II/LC3-1 ratio and increased p62 expression. Fianally, knockdown of Beclin1 increased the levels of bax, cleaved caspase 3 and cytochrome C and decreased bcl-2 expression. Conclusions: This study demonstrated that ALT exerts antitumor activity through inducing apoptosis and inhibiting autophagy by upregulating AP2M1 in ALL, indicating a potential therapeutic strategy for ALL treatment.

Background Acute lymphoblastic leukemia is the most common leukemia characterized by uncontrolled proliferation of immature lymphoid cells [1,2]. Over the decades, Despite signi cant advances to make in the treatment of ALL, about 25% of children and half of the adults are still not sensitive to chemotherapy or relapse [3][4][5]. So far, the available treatment options for ALL include chemotherapy, antibody therapy and allogeneic bone marrow transplantation depending upon the stage of cancer. Antibody therapy such as anti-CCR4, daclizumab, and alemtuzumab, combined treatment with AZT and IFN, and allogeneic bone marrow transplantation have been suggested to cure ALL. Unfortunately, due to the various limitations, the ideal effect has not yet been achieved [6].
ALT, a major bioactive sesquiterpene component of Inula helenium, has been reported to possess multiple biological and pharmacological activities including antibacterial, antifungal, antiin ammatory and anticancer effects [7]. In recent years, ALT has been reported to exert antitumor effect on many cancers, including lung cancer, gastric cancer, hepatic cancer, B-cell acute lymphoblastic leukemia, pancreatic cancer and breast cancer [8]. Moreover, ALT has been shown to have synergistic antitumor effect with other medicines. For example, Wang J et al. reported that ALT could enhance the sensitivity of lung cancer cells to gemcitabine [9]. Cao et al. stated that ALT might improve the therapeutic e ciency of chemotherapy drug oxaliplatin [10]. Zheng et al. demonstrated that ALT could sensitize human pancreatic cancer cells to EGFR inhibitors [11]. Further, ALT exerts antitumor activities through a variety of molecular mechanisms. Firstly, ALT promotes ROS-mediated inhibition of Akt/glycogen synthase kinase (GSK)3β pathway and induction of endoplasmic reticulum (ER) stress [9]. Secondly, ALT regulates p38 MAPK and NF-κB pathways. Thirdly, ALT can inhibit TrxR1 activity and activate ROS-mediated p38 MAPK pathway [12]. Fourthly, ALT impairs autophagy-lysosome pathway via targeting TFEB [12]. Finally, ALT enhances the sensitivity of cancer cells to EGFR inhibitors through inhibiting STAT3 signaling [11]. These pathways are overlapping and interact with each other. However, the role and molecular mechanism of ALT in ALL remain unexplored.
AP2M1 encodes the μ-subunit of the adaptor protein complex 2 (AP-2), which belongs to the adaptor complexes medium subunits family. AP2M1 was initially identi ed to be involved in clathrin-mediated endocytosis and intracellular tra cking. AP2M1 is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit stimulates clathrin and supports the cell surface receptor incorporation. AP2M1 is required for hepatitia C virus, rabies virus and dengue virus infection, and AP2M1 knockdown reduces viral titer or infectivity [13][14][15]. Moreover, abnormal AP2M1 causes developmental and epileptic encephalopathy through impairing clathrinmediated endocytosis [16]. In addition, AP2M1 is involved in the transmission of secreted signals produced by senescent cells [17]. Recently, AP2M1 has been found to be associated with cancers. Cong-Cong Wu et al. demonstrated that the expression of AP2M1 was signi cantly elevated in adenoid cystic carcinoma and mucoepidermoid carcinoma and closely correlated with the proliferation marker cyclin D1 [18]. AP2M1 can also serve as a prognostic marker of hepatocellular carcinoma. AP2M1, as a transcription factor, speci cally binds to the hepatocyte growth factor gene promoter to repress its activity [19,20]. All these ndings indicate diverse roles of AP2M1 in human diseases.
In this study, we rstly demonstrated that ALT inhibited the proliferation of ALL cells in does-dependent manner. Importantly, AP2M1 was identi ed to mediate the antitumor effect of ALT on BV173 and NALM6 cell lines. Subsequently, the relationship between autophagy and apoptosis was evaluated by administration of rapamycin. Finally, the effect of overexpressed AP2M1 on autophagy and the effect of Beclin1 knockdown on apoptosis were examined.

Methods And Materials
Cell culture and reagents The human acute lymphoblastic leukemia cell lines (BV173, NALM6, JM-1, NALM1, RS6 and SUPB15) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) . All cell lines were cultured in Dulbecco's altered Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA) at 37℃ with 5% CO 2 in a humidi ed incubator. Alantolactone and rapamycin were obtained from MedChemExpress (New Jersey, USA). Both the tested compounds were dissolved in dimethylsulfoxide (DMSO). The nal concentration of DMSO in culture medium did not exceed 0.1%.

Cell viability assay
Cell viability assay was evacarried out with a MTT assay kit (Sigma-Aldrich, USA). BV173 and NALM6 cells were seeded in 96-well plates and then treated with 1, 5, and 10 μM of ALT for 24h. After that, 10μl of 5 mg/ml MTT solution was added, and incubated at 37 o C for 2 h. MTT solvent was then added to the cuture. The absorbance was read at 570 nm using a microplate reader. Each experiment was performed at least three times.

RNA sequencing
All of these procedures were performed by the Beijing Genomics Institute (BGI, Shenzhen, China). In brief, BV173 and NALM6 cells were treated with ALT (5μM) and control (0.1% DMSO) for 24 hours. JC-1 staining JC-1 staining was performed to assess the mitochondrial membrane potential (MMP) using a JC-1 assay kit (Beyotime, Shanghai, China) according to the operating instruction. Images were taken under a uorescence microscope (Leica, Wetzlar, Germany). The ratio (%) of red/green uorescence intensity was calculated by Image J software.
Immuno uorescence BV173 and NALM6 cells were re-suspended to prepare cell suspension after treatment with the designated reagents. 40ul of cell suspension was dropped on coverslips. After that, the coverslips covered with cells were dried in an oven at 50℃, and then washed with PBS. Next, cell coverslips were xed in 4% paraformaldehyde for 15min, and permeabilized with 0.2% Triton X-100 for 15 min. Subsequently, the cells were sealed with 5% BSA solution at 37 °C for 40 min, and then incubated with anti-AP2M1(Shanghai, China) primary antibodies at 37°C for 4 h.The cells were incubated with the Alexa Fluor®594 or 488 secondary antibodies (Abcam, Shanghai, China) at 37°C for 1 h and stained with DAPI for 3min. Finally, antifade mounting medium was placed on the slide and covered with a cell-coated glass sheet. The glass slides were observed by a uorescence microscope (Leica, Wetzlar, Germany). Image J software 2.1 was used to measure the uorescence intensity.

Confocal analysis
For confocal microscopy, the GFP-LC3 vector was transfected into the BV173 cells to generate a stable GFP-LC3 BV173 cell line. After treatment indicated, the GFP-LC3 BV173 cells were xed in 4% paraformaldehyde for 15min. Subsequently, the cells were permeabilized using 0.05% TritonX-100, and stained with DAPI (Invitrogen, Eugene, OR, USA). In the end, the cells were examined and quanti ed using the AOBS confocal laser scanning (Leica, Wetzlar, Germany).

Xenograft model
Experiments were performed in BALB/c nu/nu mice (6 weeks old), which were obtained from Charles River Laboratories. A number of 3×10 6 BV173 cells transfected with AP2M1 siRNA in 100 μl PBS were subcutaneously injected into the posterior ank region of nude mice. The long diameter and short diameter of tumor were measured every 2 days, and calculated using the formula as follows: tumor volume = 0.5 × long diameter × short diameter 2 . The mice were treated with ALT every day after injection 15 days. Finally, the mice were sacri ced and excised the tumors.
Statistical analysis SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) were used to analyze all data for statistical signi cance. All the variables were presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey's Post hoc test was used to assess the difference between multiple groups. Differences between two groups were analyzed by the Student's t-test. P<0.05 was considered as statistical signi cance.

ALT inhibites the proliferation of acute lymphoblastic leukemia cells
To investigate the potential effect of ALT on ALL, ALL cell lines, including BV173, JM-1, NALM1, NALM6, RS6, and SUPB1, were treated with 1, 5 and 10μM of ALT for 24 h. Cell viability was determined by MTT assay. The results showed that both 5 and 10 μM of ALT signi cantly inhibited the proliferation of BV173 ( Figure 1A), NALM1( Figure 1D), NALM6 ( Figure 1C), and RS6 cells ( Figure 1E), while 10μM of ALT inhibited the proliferation of JM-1 ( Figure 1B) and SUPB1 cells ( Figure 1F). 5 μM of ALT exerted signi cant growth inhibitory effect on BV173 and NALM6 cell lines, and thus was chosen for further experiments.
ALT promotes the expression of AP2M1 To elucidate the mechanism underlying the effect of ALT on ALL cells, we screened the expression pro le of mRNA using RNA-seq. As shown by the heatmap (Figure 2A), AP2M1 was notably upregulated in both BV173 and NALM6 cells treated with 5μM of ALT. To con rm this result, qRT-PCR and western blot were performed to detect AP2M1 expression in BV173 and NALM6 cells. As expected, a dose-dependent increase of AP2M1 mRNA and protein expression was observed in ALT-exposed BV173 and NALM6 cells ( Figure 2B-D). Further, cell immuno uorescence assay visually demonstrated that ALT promoted the increase of AP2M1 protein in a dose-dependent manner ( Figure 2E). Accordingly, these results indicate that ALT treatment induces the expression of AP2M1 in ALL cell lines.
ALT inhibites proliferation and colony formation of ALL cells by targeting AP2M1 To check out whether AP2M1 is involved in the cytotoxicity of ALT, BV173 and NALM6 cells were cotreated with ALT and si-AP2M1. As shown in Figure 3A, ALT upregulated AP2M1 expression while the transduction of AP2M1 siRNA signi cantly reduced AP2M1 expression, indicating AP2M1 siRNA could effectively inhibit the expression of AP2M1 in BV173 and NALM6 cells. Subsequently, the effect of ALT and si-AP2M1 on cell proliferation was assessed using MTT and colony formation assays. MTT assay showed that ALT inhibited cell viability of cells, while AP2M1 knockdown reversed the toxic effect of ALT, indicating that AP2M1 was involved in the toxic effect of ALT on cells ( Figure 3B). Consistent with the results from MTT assay, the colony formation assay also showed the same conclusion ( Figure 3C). Collective data demonstrate that ALT exerts growth inhibitory effect in ALL cells via upregulating AP2M1.

ALT inhibites the acute lymphoblastic leukemia growth in vivo
Based on these ndings, xenograft model experiment was further conducted to investigate the impact of ALT on ALL. As shown in Figure 4 B, from day 14, tumor volume in the ALT group was signi cantly lower than that in the control group, whereas xenograft tumor volume in cotreatment group with ALT and si-AP2M1 was larger than that in the ALT alone group (p < 0.05). The xenograft tumors were weighed after the mice were sacri ced. As shown in Figure 4A and 4C, tumor weight showed the same conclusion. Accordingly, these results indicate ALT suppresses the tumor growth in vivo and this effect could be reversed by knockdown of AP2M1.

ALT induces apoptosis and inhibites autophagy of ALL cells via upregulating AP2M1
The mitochondrial membrane potential (MMP) is an important indicator of cell function and health, and its dissipation is considered as an early indicator of cell apoptosis. To evaluate the effect of ALT on cell apoptosis, ow cytometry, JC-1 staining and western blot were used to measure cell apoptotic rate, MMP and apoptosis-related proteins, respectively. As showed in Figure 5A, treatment with 5μM of ALT for 24 h dramatically increased the apoptotic rate of BV173 and NALM6 cells, while AP2M1 knockdown remarkably diminished this pro-apoptotic effect of ALT. Meanwhile, JC-1 staining showed that ALT signi cantly decreased the MMP of ALL cells, which was reversed by AP2M1 knockdown ( Figure 5B). Furthermore, western blot showed that ALT treatment substantially elevated the levels of cleaved caspase-3, bax and cyt-c in cytoplasma while decreased bcl-2 expression, but these effects were attenuated by AP2M1 siRNA ( Figure 5E). These results indicate that knockdown of AP2M1 attenuates the pro-apoptotic effect of ALT via maintaining mitochondrial function and inhibiting caspase cascade.
Next, we detected the autophagy of BV173 and NALM6 cells by immuno uorescence staining and western blot. Immuno uorescence assay results revealed that ALT treatment reduced the number of the LC3 uorescent puncta compared to the control group, while AP2M1 knockdown increased the number of LC3 puncta in comparison to the ATL group ( Figure 5C). Besides, western blot analysis demonstrated that ALT signi cantly decreased the expression of autophagy markers Beclin1 and LC3II/LC3I ratio while increased the expression of autophagy substrate p62 protein. However, ALT-mediated regulation of these autophagy-related proteins were reversed by AP2M1 knockdown, indicating the involvement of AP2M1 in ALT-mediated autophagy regulation ( Figure 5D). Above all data support the notion that ALT induced apoptosis and inhibited autophagy of ALL cells via upregulating AP2M1.
Rapamycin-induced autophagy attenuates the pro-apoptotic effect of ALT Autophagy and apoptosis are necessary to maintain the cellular homeostasis. There is a complex relationship between autophagy and apoptosis. In general, autophagy signaling can prevents the induction of apoptosis, and apoptosis-related caspase activation, as a feedback response, can inhibit the autophagic process. However, autophagy can also trigger apoptosis under certain cases. To further explore the relationship between autophagy and apoptosis induced by ALT, BV173 and NALM6 cells were treated with ALT alone or combination with autophagy activator rapamycin. Immuno uorescence assay results revealed that ALT treatment reduced the number of the LC3 uorescent puncta compared to the control group, while rapamycin increased the number of LC3 puncta in comparison to the ATL group ( Figure 6C). Western blot analysis showed that apamycin could reverse down-regulated Beclin1 and LC3II/LC3I ratio and elevated p62 level induced by ALT in BV173 and NALM6 cells ( Figure 6D), suggesting that rapamycin could e ciently activate autophagy. Further, the effect of autophagy activator rapamycin on pro-apoptotic activity of ALT was evaluated. The results showed that rapamycin could partially abolish increased apoptotic rate and MMP caused by ALT, as demonstrated in Figure 6A-B. Moreover, the nding was con rmed by expression pattern of apoptosis related-proteins ( Figure 6E). These ndings indicate that ALT could induce cell apoptosis via inhibting autophagy in ALL.

AP2M1 inhibites autophagy and induces apoptosis
As AP2M1 is essential for ALT-mediated apoptosis and autophagy of ALL cells, we next examined whether overexpressed AP2M1 affects apoptosis and autophagy. Western blot assay demonstrated that overexpressed AP2M1 inhibited the expression of autophagy-related markers Beclin1 and LC3II and increased the expression of p62 ( Figure 7A). Moreover, the knockdown of Beclin1 caused a signi cant increase of apoptosis-related markers such as bax, c-cyt-c and cleaved caspase 3 and a obvious reduction of bcl-2, indicating that Beclin1 affects the process of cell apoptosis. Taken together, the present study suggest that ALT may maintain cellular homeostasis between autophagy and apoptosis by increasing AP2M1, and thereby exert anti-tumor activity in ALL ( Figure 7C).

Discussion
In this study, ALT was found to promote apoptosis of acute lymphotic leukemia cells by inhibiting autophagy. In total, two novelties were made in this study. Firstly, this study provided evidence that ALT exerted an anti-tumor activity via regulating autophagy and apoptosis of ALL cells. More important, this study has shown that AP2M1 protein contributed to the anti-tumor effect of ALT on cancer cells by modulating autophagy.
There are many types of research that show that ALT exerts anti-tumor effects on tumor cells through alone or combined treatment with other anti-tumor agents The mechanisms included regulation of oxygen species-mediated ER stress, ROS response, and other signal pathways such as Akt/GSK3β pathway, p38 MAPK, NF-κB pathway, STAT3 signaling, and Nrf2 signaling. Currently, the function of ALT on autophagy was reported only once in which He et al. found that ALT caused the accumulation of autophagosomes due to impaired autophagic degradation and signi cantly inhibited the activity and expression of CTSB/CTSD proteins [12]. Their data demonstrated that ALT, which impaired autophagic degradation, was a pharmacological inhibitor of autophagy in pancreatic cancer cells and markedly enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin. In this study, we demonstrated that inhibition of autophagy was also the mechanism of ALT to suppress the growth of ALL and this result contributed new proof to the involvement of autophagy in the anti-tumor effects of ALT.
ALT has been shown to contribute to cell apoptosis in numerous cancers. For example, ALT induces gastric cancer BGC-823 cell apoptosis by regulating the AKT signaling pathway [21]. ALT induces apoptosis of breast cancer cells via the p38 MAPK, NFκB, and Nrf2 signaling [22]. ALT also induces apoptosis and enhances the chemosensitivity of A549 lung adenocarcinoma cells to doxorubicin [23]. In this research, it was indicated that ALT also promotes the apoptosis of ALL cells.
Moreover, as suggested in the ndings, ALT signi cantly stimulated apoptosis but at the same time inhibited autophagy of BV173 and NALM6 cells. This function was further con rmed by the use of rapamycin. The results showed that rapamycin signi cantly induced autophagy and reversed the effect of ALT on apoptosis, indicating ALT induces apoptosis partially through inhibiting autophagy. Necrosis, apoptosis, and autophagy are three types of programmed cell death that contribute critically to cancer cell progression, division, and metastasis [23,24]. However, the relationship between autophagy and apoptosis remains complicated. In some cases, autophagy promotes cell apoptosis. Paris saponininduced autophagy promotes acute lymphoblastic leukemia cell apoptosis through the Akt/mTOR signaling pathway [25]. Parthenolide inhibits pancreatic cell progression by autophagy-mediated apoptosis [26]. In other cases, autophagy inhibited the apoptosis process. For example, Rottlerinstimulated autophagy results in apoptosis in bladder cancer cells [27]. Autophagy inhibition improves heat-stimulated apoptosis in human non-small cell lung cancer cells through ER stress pathways [28].
In order to nd out the mechanism underlying the effect of ALT on apoptosis and autophagy, We tried to identify the key factors involved in both apoptosis and autophagy. In this study, AP2M1 was found to be upregulated in ALL cells treated with ALT. Further, AP2M1 knockdown signi cantly inhbited the effect of ALT on apoptosis and autophagy, while AP2M1 overexpression inhibited autophagy and induced apoptosis. These ndings indicate AP2M1 plays an important role in antitumor activity of ALT. Some studies have shown that AP2M1 is related to the occurrence and development of cancers, including medulloblastoma, head and neck squamous cell carcinomas, esophageal squamous cell carcinoma,chronic myeloid leukemia, prostate cancer, and HCC [8,19,20,29,30]. AP2M1 is identi ed to function in clathrin-mediated endocytosis and intracellular tra cking. However, the role and mechanism of AP2M1 in tumor remains are still not fully understood. Meisel Sharon S et al. showed that AP2M1 is associated with transcription factor ERG in cell nuclei, and plays a mechanistic role in insulin-like growth factor-1 receptor internalization [20]. AP2M1 is also essential for recruitment of receptor tyrosine kinases (RTKs) and components of signal transduction cascades to chromatin encompassing transcribed genes [31]. In present study, we found AP2M1 not only regulated the expression of autophagy-related markers Beclin 1, LC3, and p62, but also modulated the expression of apoptosis-related markers bcl-2, bax and cleaved caspase 3. These data indicate that AP2M1 acts as a upstream signal molecule to regulate cell apoptosis and autophagy. Therefore, based on relevant research and our ndings, we speculate that upon ALT treatment, AP2M1 may promote some kinases internalization and coordinate genome-wide transcriptional events to regulate apoptosis and autophagy. These assumptions has not been validated and need more experiments for further research.

Conclusion
Taken together, we showed that ALT was able to prevent the proliferation of acute lymphoblastic leukemia cells by inducing apoptosis and inhibiting autophagy. The underlying mechanism was involved in the regulation of the AP2M1.

Declarations
Authors' contributions DY, JW, ZL and YL designed the experiments, performed the research and analyzed the data. YT, WL and MG conducted the cell culture and western blot. TY, FC and JZ did literature research and data analysis. DY and JW wrote and revised the manuscript. YL offered professional advices about the whole research. All authors read and approved the nal manuscript.
The datasets during and/or analyzed during the present study available from the corresponding author on reasonable request.

Consent for publication
Not applicable.

Ethics approval and consent to participate
The study were approved by the Committee for Animal Experimentation of the First A liated Hospital of Harbin Medical University. The animal study was complied with the guiding principles for the care and use of laboratory animals. All protocols and methods were in accordance with the guidelines and regulations.