Erianin suppresses the development of acute myeloid leukemia via PPARɑ and regulating PI3K/AKT signaling pathways

doi:10.21203/rs.3.rs-5279493/v1

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Erianin suppresses the development of acute myeloid leukemia via PPARɑ and regulating PI3K/AKT signaling pathways

https://doi.org/10.21203/rs.3.rs-5279493/v1

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Acute myelogenous leukemia is a highly invasiveness hematological malignancies that mainly relies on chemotherapy to improve survival rate at present.Up to now,the application of natural products has been widely accepted as an alternative compared with conventional chemotherapeutic agents due to their relative safety.In our research,we attempted to explore the anti-AML capacity and the potential mechanism of Erianin(Eri), a natural component from Chinese herbal medicine Dendrobium flaviflorum.We verified that Erianin inhibited the proliferation of AML cells and induced their apoptosis.Erianin can block cell cycle of AML cells at G2/M phase thourgh regulation of cell cycle-related proteins and P21,P27,P53 mRNA expression.Mechanistically, we firstly filtered and locked PPARɑ and PIK3R1 through network pharmacology analysis, protein-protein interaction (PPI) network, GO and KEGG pathway enrichment analysis, and further confirmed their good binding with Erianin by molecular docking.Specifically,Erianin inhibited the transcriptional level of PIK3R1 by activating the protein level of PPARɑ, thereby inhibiting the PI3K/AKT pathway.The inhibitory ability of Erianin on AML cells was partially neutralized by GW6471,an inhibitor of PPARɑ.It was also worth noting that Erianin revealed vigoroso coordinate repression with LY294002 on AML cells. Overall, these evidences indicated that Erianin exerted an influence on AML cells at least partially through PPARɑ to regulate PI3K/AKT signaling pathways.In summarize, we demonstrated the potent anti-AML effect and the potential mechanisms of Erianin, which suggested a hopeful strategy that Erianin has the capacity to be developed into a novel anti-AML drug.

acute myeloid leukemia

peroxisome proliferator-activated receptors

Phosphatidylinositol 3-Kinase

erianin

Acute myelogenous leukemia (AML) is an aggressive clonal malignant hematological illness characterized by a speedy recrudescence and muti-drug resistance.AML usually damages bone marrow hematopoiesis and causes unnecessary peripheral blood hemocytopenia.Up to now, AML patients used to have restricted therapy choices, subsisted mainly on cytarabine + anthracycline (7 + 3) intensive induction chemotherapy[1].The median age of diagnosed patients at diagnosis is 70 years ,prognoses have always been inconsistent and unfavourable. The 5-year overall survival rate is about 35% for younger patients and 15% for patients over 60 years of age[2, 3].Therefore, it is particularly vital to further study the nosogenesis of AML and explore new diagnosis targets,which contributes to achieve early diagnosis and improves the overall survival of AML.

The application of natural products has been widely accepted as an option for the treatment of cancer due to their relative safety compared with conventional chemotherapeutic agents. It has been proved that many drugs from natural plants can act on multiple targets and improve the overall health of patients from multiple mechanisms at the same time, and have lower drug resistance and toxicity compared with drugs in the same field, which is more conducive to the long-term use of patients[4,5].In fact,in the field of cancer,most drugs actually being either natural products or directly derived therefrom[6].As a natural biphenyl compound in Dendrobium, Eri is one of the crutial pharmacological ingredient in Dendrobium[12].Its pharmacological functions including anti-tumor[7],immunomodulatory[7], anti-inflammatory[8], anti-angiogenic[9], antibacterial action and antivirus activity[10, 11].In the aspects of anti-cancer, Eri exerts various degrees of inhibition on cancers that exists basically every part of the body through different mechanisms,such as drug resistance, cell cycle arrest, and pro-apoptosis or anti-proliferation of cancer cells[12].Until now,research on the anti-cancer effects of erianin is limited.A study by Wang, Yan et al reported that Erianin inhibited the development of PLGC by regulating the HRAS-PI3K-AKT signaling pathway[13].It is further demonstrated that Erianin induced cell apoptosis, G2/M arrest, and GSDME-dependent pyroptosis through reducing the expression of PPT1 and phosphorylation of mTOR in OSCC cells[14].More importantly,two studies convincingly evidenced that Eri can respectively inhibit tumor development from novel mechanisms and traditional signal transduction pathways

Eri exerts anti-tumor ability by triggering Ca2+/CaM-dependent ferroptosis and inhibiting cell migration[15],and as a novel inhibitor of CRAF and MEK1/2 kinases, Eri suppress the MAPK signaling pathway which is activated by BRAF V600E or RAS mutations in Melanoma and colorectal cancer[16].Taken together,above results summarily suggested that Eri could be considered as a novel anti-cancer Ingredient.Regrettably,in addition to these studies ,no further research has been performed to discuss whether Eri can exert the same anti-tumor effect on acute myeloid leukemia and in the area of AML,it is a necessity to deeply explore the specific molecular mechanism underlying the anti-cancer effects of Eri.

To date,despite it has been clarified that Eri induced apoptosis in HL-60 cells by Li, Y M et al[17],the specific mechanisms of how Eri regulates cell death in AML are still remain unknown.The purpose of our research was to confirm whether Eri also had the same coordinate repression on other AML cell lines, and to explore the potential mechanism.To be specific,we found that Eri has a certain coordinate repression on all AML cell lines by regulating PI3K/AKT signaling pathways.Through utilizing network pharmacology and molecular docking, we also found that Eri plays a role mainly by regulating the expression level of PPARɑ protein,thus inhibits the mRNA expression of PIK3R1 to refain PI3K/AKT pathways.More importantly,it is surprised that the combination of Eri and PI3K inhibitor LY294002 shown a synergistic inhibitory effect on AML cells, which provides a noval treament thought for Eri as an anti-tumor drug .

Cell lines and culture

The AML cell lines used in our study were THP-1, KG-1a, U937, NB4, and HL-60.All of them were available in our laboratory.The cells were cultivated in RPMI-1640 medium contained with 10% FBS and incubated at 37 C under 5% CO2 atmosphere.

Chemicals and reagents

Erianin(HY-N0571), GW6471(HY-15372), LY294002(HY-10108) were obtained from Med Chem Express (MCE, NJ, USA).RPMI-1640 medium was obtained from Gibco (MA, USA).The cell counting kit-8 (HY-K0301) was purchased from Solarbio (Beijing, China).

Cell proliferation and viability assays

The CCK8 assay was uesd to monitor the proliferation rates of cells and the inhibition rates of cells.In brief,1×10⁴ cells/well were seeded in 96-well plates averagely and incubated with Eri on a concentration of 0, 1, 3.3, 10,33,100,333,1000,3333 nm, then added 10µl CCK8 at 0,24,48,72 h to terminate the operation.The Optical density (OD) value detection were performed after incubating for 2h and was recorded at 450nm by microplate reader. 1 × 10⁴ KG-1α or THP-1 cells were totally inoculated in 96-well plates which contain 100 µL medium and then treated with DMSO, the appropriate doses of Eri and GW6471/LY294002 solely or in combination treat for 24 h.Ultimately, the CCK-8 kit was conducted to detect cell viability and then converted to inhibition rates.

Cellular apoptosis detection

To evaluate the cell apoptosis,the particular cells were inoculated on 6-well plates at a density of 3 × 10⁵ cells / well and treated with indicated agentia.After the treatment,the cells were gathered and washed three times with PBS before detection.The apoptosis rate was detected by the Annexin V-FITC Apoptosis Detection Kit (SUNGENE BIOTECH, Tianjing) and the folw cytometric analysis was operated by a CytoFLEX flow cytometer (Beckman Coulter, USA).

Cell cycle analysis

To evaluate the cell cycle analysis, the cells were collected after being treated with Erianin and washed three times with PBS.For the detection, 1 × 10⁶ cells were treated with 75% ethanol and treated overnight at 4℃,then incubated with propidium iodide (PI; 100 mg/ml) for 30 min at room temperature.The BD Biosciences FACSCaliburTM flow cytometer were operating to analyze the cell cycle phases.

Network pharmacology analysis

The 2D structure file of Eri was obtained from Pubchem database and imported into PharmMapper database to predict Eri related potential targets.Using “Acute Myelogenous Leukemia”as keyword to seek for AML-related targets in GeneCards database and utillizing online Venn diagram to receive interactive targets of Eri and AML.Established by STRING database,the protein-protein interaction (PPI) network was ultimately visualized by Cytoscape software to acquire interactive targets.

Molecular docking

As previously described,obtaining the 2D structure of Eri using the Pubchem Database and converting it to 3D form by Openbabel.Next, aquiring the 3D construction of the target from the PDB protein database .The docking and binding energy of the target molecule and Eri were assessed by Autodock Vina.Conducted models by AutoDock Vina software, the results of molecular docking simulation which contained molecular bonds and bond energies for docking were ultimately visualized by Pymol software.

Western Blotting

After treating with the indicated reagent, the cells were washed three times with PBS and dissolved with PMSF(Beyotime, China) combined with RIPA buffer (Beyotime, China) at a ratio of 1:100.The BCA protein assay kit (Beyotime, China) was used to extract and assesse the concentration of proteins .Briefly, the protein were mixed with 5 × loading buffer and then heated for 10 min.Using Polyacrylamide gel electrophoresis(PAGE) to get protein seperated and then electrotransferred onto a PVDF membrane (Millipore, USA).The membranes were incubated with the indicated primary antibodies at 4°C for 17 h after blocking with a 5% skimmed milk powder.Wait for one night,incubating with relevant secondary antibodies for1h at room temperature, the membranes were ultimately analysed with enhanced chemiluminescence (ECL). The used primary antibodies in our study were as follows :PPARɑ ,PARP,Cleaved-PARP ,Caspase3, Cleaved-Caspase3, P-PI3K, PI3K, AKT,P-AKT,CylinB1,CyclinA2(CellSignalingTechnology,USA);P85ɑ(abcam,China);Bcl-2,Bax ,P27(ZENBIO,China);CDK1(Wanlei,China);GAPDH(Proteintech,USA).

Quantitative real‑time PCR

The TRIzol reagent (Takara, Japan) was uesd to isolated RNA and transcribed them to cDNA by conducting the PrimeScript™ RT reagent kit (Takara).RT-qPCR was operated by CFX Connect™ RT-qPCR system (Bio-Rad, USA) by SYBR® Premix Ex Taq™ II kit (Takara). .

All primers were obtained from Sangon Biotech (Shanghai, China):

The HS-ACTB primers:(Forward:5′-CCTGGCACCCAGCACAAT-3′,Reverse:5′-GGGCCGGACTCGTCATAC-3′);

The PPARɑ

primers:(Forward:5′-AGATTTCGCAATCCATCGGC-3′,Reverse:5′-GCGTGGACTCCGTAATGATA-3′);

The PIK3R1 primers:(Forward:5′-TAGCTCGCGCGATCTAGGGGC-3′,Reverse:5′-CGCGATCAATAAAGCTAG-3′);

The P21 primers:(Forward:5′-AGCGATGGAACTTCGACTTTG-3′,Reverse:5-CGAAGTCACCCTCCAGTGGT-3′);

The P27 primers:(Forward:5′-ATCACAAACCCCTAGAGGGCA-3′,Reverse:5′-GGGTCTGTAGTAGAACTCGGG-3′);

The P53 primers:(Forward:5′-AAAGTGCGTCCGTTCTCAATG-3′,Reverse:5-GGTTCTTCCTCAGAGTACCAAAG-3′)

Statistical analysis

Operating for at least three independent experiments,all data were presented as mean ± standard deviation (SD).Unpaired Student’s t testsone-way or two-way analysis of variance (ANOVA) was perfromed by the GraphPad Prism8 software. P-value less than 0.05 was considered as statistically significant.P-value < 0.05, < 0.01, < 0.001, < 0.0001 respectively represents a statistical significance by “*”,“**”, “***” and “****”.

Erianin inhibits the proliferation and induce apoptosis of AML cells in a dose-dependent manner at low sodium concentration

As a compound extracted from the Chinese herbal medicine Dendrobium flaviflorum,Eri’s chemical 2D structure was shown in Fig. 1A.To explore the anti-tumor effect of Eri on AML cells,we examined the cell viability of each AML cell line(THP-1、U937、NB4、HL-60、KG-1a) after 24 hours of Eri treatment by CCK-8 cell proliferation assay(Fig. 1B).The results shown a remarkable anti-tumor effect on AML cells performed by Eri,we chose KG-1a cell line which was most sensitive to Eri and THP-1 cell line which was relatively resistant to Eri for further experiments.In the same way,CCK-8 assay also confirmed the inhibitory effect of Eri which can inhibit the proliferation of THP-1 and KG-1a cells in a time gradient and dose concentration gradient(Fig. 1C).Furthermore,we tempt to clarify whether Eri induced the apoptosis of AML cells.The results of flow cytometry analysis were shown in Fig. 1D and Fig. 1E,after 48h treatment with Eri,the apoptotic rates of THP-1 and KG-1a cells was remarkably enhanced in a dose-dependent pattern. Next, we performed western blot to evaluate the expression of apoptosis proteins after Eri’s treatment and discovered that the expression of pro-apoptotic proteins including Cleaved-Caspase3 and Cleaved-PARP, which served as recognized markers of apoptosis, were enhanced after Eri’s reatment(Fig. 1F).Moreover,we discovered a noteworthy enhancement of pro-apoptotic factor Bax,however, an obvious reduction of anti-apoptotic factor Bcl-2 in AML cells treated by Eri(Fig. 1F).To sum up,these evidence suggest that Eri mainly induce the apoptosis of AMLcells through adjusting the expression of Bcl-2 and Bax.

Erianin induces G2/M phase arrest in AML cells

As shown in Fig. 2A,B ,we verified that Eri could induce G2/M phase cell cycle arrest by flow cytometry.It is reported that activation of CDK1 and Cyclin B1 is necessary for the progression of G2/M whereas CDKIs and p27 suppress cell cycle progression[38].According to the results shown in Fig. 2C,the expressions of p27 and Cyclin B1 were up-regulated, whereas the expressions of CDK1 was significant down-regulated.It was worth noting that Eri also partially up-regulated the expression of CyclinA2,which suggested that Eri can also cause cell cycle arrest in S phase to a certain extent(Fig. 2C).Meanwhile, the mRNA expression of P27,which is as same as the Western blot result, was up-regulated due to the results of Q-PCR(Fig. 2E).In the same way,after treating with Eri for 24h,the mRNA expression of P21,P53 also shown various degrees of increase(Fig. 2D,F).In summary,the above evidences indicate that Eri can induce cell cycle arrest by regulating G2/M phase related cell cycle proteins and up-regulating p21,p27,p53 mRNA expression to control cell cycle of AML cells.

Screening the target genes and pathways of Erianin's anti-AML activity by network pharmacology

To explore the mechanism underlying Eri anti-tumor ability, we conducted network pharmacological analysis to get possible targets of Eri acts on AML.With collecting 10528 specific AML-related targets and 31 unique Eri-related targets, we screened a total of 258 cross targets of them by online Venn diagram(Fig. 3A).Based on these interaction targets, we created a protein-protein interaction (PPI) network to detect the mutual regulation between the intersecting targets(Fig. 3B).On the basis of PPI network, the top 20 hub protein targets were chosen and arranged by BC value with a brief message(Fig. 3C).Meanwhile, after comprehensive evaluation,the top 10 protein targets networks are arranged according to the size of the MCC value(Fig. 3D).As shown in Fig. 3C,we validated them one by one in order of scores and based on the currently ongoing study, finally confirmed that PPARɑ may be the main target for Eri to exert anti-AML effects.On the other hand,as one of the top hub target and the major regulatory subunit of PI3K,PIK3R1 particularly draw our attention(Fig. 3D,F).

To further confirm,we conducted molecular docking simulation Vina to estimate the potential union of Eri to PPARɑ(PDB:2P54)) and PIK3R1(PDB:315R )by AutoDock .The results turned out that the free binding energy after caculating of Eri to PPARɑ and PIK3R1 was 3.2kcal/mol and 2.4kcal/mol, suggesting a relatively ideal binding affinity of Eri to both of them.As the presented binding results shown that the PPARɑ protein binds Eri at the Gly-439 residue, while the PIK3R1 protein binds Eri at the Val-32 residue(Fig. 3E).To predict the potential anti-AML target pathway of Eri, we conducted KEGG and GO enrichment analysis for cross targets of AML and Eri .According to the top 20 results from GO enrichment analysis, the corporate targets are mostly related to phosphorylation,proteolysis,hormone-mediated signaling pathway and so on(Fig. 3G).Due to the result of KEGG enrichment analysis,it is noteworthy that those cross-targeted were significantly enriched in PI3K-AKT pathway (Fig. 3G).

Erianin exploits anti-AML effect through PI3K/AKT signaling pathways

Furthermore, we conducted western blot to determine whether Eri could activate PPARɑ protein and regulate PI3K-AKT pathway at the same time.We found that Eri prominently decreased the expression of PI3K protein, the phosphorylation of PI3K as well as the level of AKT protein, the phosphorylation of AKT,which comfirmed that Eri mainly plays an anti-AML effect by inhibiting the PI3K-AKT pathway.In addition,Eri also enhanced the expression of PPARɑ in a dose-dependent way,which further supports our hypothesis(Fig. 4A).In order to verify from the opposite direction,

Using the PI3K inhibitor LY294002 in combination with Eri to observe the effect on cell proliferation, the result turns out that they can synergistically inhibit the proliferation of AML cells with a concentration gradient (Fig. 4B).As it was shown in Fig. 4C, the synergy index between Eri and LY294002 was between 0.1 and 0.3, indicating that they had a strong synergistic effect in reducing the viability of KG-1α and THP-1 cells.To further confirm the combination effect between two of them,we conducted flow cytometry to detect apoptosis rates of THP-1 and KG-1a cells after the combination,the results also show a strong synergistic effect in promoting apoptosis in AML cells(Fig. 4D,E).

Erianin-mediated PPARα activation down-regulates PIK3R1 to regulate PI3K/AKT pathway

Nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is a crutial ingredient related to regulate energy metabolism,mitochondrial and peroxisomal function,as a therapeutic target for CLL,it also has been recognized as a novel target for eliminating stem and progenitor cells in AML[18–20].To further verify whether PPARɑ is the main target of Eri in AML cells,we used PPARɑ inhibitor GW6471 and evidenced that the inhibitory capacity Eri-exert on the proliferation of AML cells was partially neutralized (Fig. 5A).Due to the result of western blot,compared to the single control group,we noticed that the the levels of pro-apoptotic proteins(Cle-PARP,Caspase 3,Bax) was significantly reduced whereas the anti-apoptosis protein was up-regulated to some extent after the combination of GW6471 and Eri(Fig. 5B).As the western blot results shown in Fig. 5F, we also noticed that the combination can restore the inhibitory capacity of Eri on PI3K/AKT pathway to a certain extent,which further verifed that Eri inhibited the PI3K/AKT pathway through the activation of PPARα.

PPAR controls the level of downstream genes through binding to RXR elements in the promoter region of target genes[21].We wonder if it is possible that PPARα activation can regulate the expression of PIK3R1 by interacting with its promoter region.In order to verify this hypothesis, we utilized UCSC and JASPAR bioinformatics tools and found out PIK3R1 has a PPARα binding site on the promoter region(Fig. 5C).Next,we used Q-PCR to explore the regulate relationship between two of them.As shown in Fig. 5E,although Eri did not up-reguline the mRNA expression of PPARα, it remarkably enhanced the protein expression of PPARα(Fig. 4A). Moreover, the Eri-mediated activation of PPARα was accompanied with the reduction of mRNA level of PIK3R1,which indicated that it was highly likely that Eri regulateed the transcriptional level of PIK3R1 by activating PPARɑ(Fig. 5D).This hypothesis was also verified at the protein level:the expression of P85ɑ(PIK3R1) was significantly reduced by Eri and partly restored after adding GW6471(Fig. 5F).

Natural products have been considered as appropriate origin for the process of noval therapeutic agents. Compared with conventional chemotherapy, these antitumoral inhibitors are typically more selective and have fewer adverse effects[22]. Erianin is a natural dibenzyl compound extracted from Dendrobium chrysotoxum, a Chinese herbal medicine[15].To date,abundant studies have found that Eri had widespread pharmacological capacities, including anti-tumor, anti-diabetic retinopathy,anti-inflammatory, antibacterial abilities[23–25].Moreover,Eri has shown remarkable antitumous effect on multiple type of cancers including Cervical carcinoma[26],Breast cancer[27], Liver cancer[28],Oral Cavity Cancer[29],Osteosarcoma[30],Lung cancer[31] and Bladder cancer[32].In the field of leukemia,despite the anti-tumor effect of Eri was both preliminary verified in Jurakt cells and HL-60 cells[33][34],its specific role and potential mechanisms in AML cells still remain unknown.In this study,we illustrated that Eri remarkably inhibited the proliferation of AML cells and induced apoptosis in AML cells.In mechanism,it is verified that Eri possibly played a role through activating PPARɑ to inactivate PI3K/AKT pathway.More surprisingly, the combination of Eri and PI3K inhibitor LY294002 exerted coordinate inhibitory impacts on AML cells, which provided a hopeful strategy to treat AML.

It is reported that the capacity to prevnt tumor cells proliferation is regarded as an important evaluation indicators of anti-cancer drugs[35].In our present research,we demonstrated that Eri prominently restrained the viability of AML cells at low nm concentrations in dose-dependent manner,and dramatically induced apoptosis of AML cells. Since apoptosis and proliferation are connected by cell cycle regulators, and cell cycle regulators can impact both cell death and cell division[36],it’s essential to explore the effect of Eri on cell cycle in AML cells.Specifically,progression into and through the cell cycle is driven by a complicated process of cell regulation, including cyclin, cyclin-dependent kinase inhibitor (CDKI) and cyclin-dependent kinase CDK[37, 38].The activation of CDK1 and Cyclin B1 is required for G2/M progression, while CDKIs,p27 negatively regulate cell cycle progression[38].In this study,we evidenced that expressions of CKI(p27) and CyclinB1 were increased, and the expressions of CDK1 was down-regulated by Eri,which was corresponding to the results of flow cytometry analysis.The induction of cell-cycle arrest is one of the earliest reported functions of p53[39].As one of the bridges connecting cell death and the cell cycle, p53 is activated to arrest the cell cycle after DNA damage, offering cells a time window to repair damaged DNA[40].Following activation of p53,p21 expression is up-regulated[41].Consistent with these conclusions,we demonstrated that Eri also activated the mRNA expression of p53 and the mRNA expression of downstream p21 mRNA expression through Q-PCR.It is konwn that the CDK inhibitor p27 is available to block cyclin A/E-CDK2 activity[41]. Due to our research,Eri could up-regulate the expression of p27 at both mRNA and protein levels,which also accorded with the above conclusion.These results suggested that a prominent G2/M phase arrest of cell cycle in Eri-treated AML cells.Moreover,we surprisingly found that Eri could arrest cells entering S phase by down-regulating the protein expression of CyclinA2 to some extent.To sum up,above results shown that Eri may exert anti-AML effects mainly through restraining cell proliferation and triggering G2/M phase arrest in AML cells.

Among the caspases, Caspase-3 is probably the most representative of apoptosis, as well as being integral to chromatin condensation and DNA breakage during apoptosis in all kind of cells[40].Specifically,when the apoptotic signal is activated, Caspase3 will be cleaved into its active form Cleaved-Caspase3,accompanied with the degradation of one of its protein substrates PARP, then triggering cell apoptosis eventually[41].Due to our current research,we evidenced that Eri initiated the intrinsic mitochondrial apoptotic pathway possibly by up-regulating the expression of Caspase3 and Cleaved-Caspase3 as well as Cleaved-PARP. B cell lymphoma 2 (BCL-2) genes regulate pro-apoptotic and anti-apoptotic intracellular signals, which are crucial for controlling programmed cell death[42].Corresponding to this,we found that Eri triggered programmed cell death by activating the level of pro-apoptotic protein Bax and inhibiting the level of anti-apoptotic protein Bcl-2. Moreover,flow cytometry results also showed that Eri exerted a potent pro-apoptotic effect on AML cells in a dose-dependent manner.Collectively, above results demonstrated that Eri is a strong apoptosis-inducing reagent to AML cells in a large extent.

To explore the potential molecular mechanisms of the anti-AML ability of Eri, we screened out some potential targets for Eri exterted on AML through network pharmacological analysis.Based on Protein-Protein Interaction(PPI) Networks,we sorted and verified one by one according to the size of nodes, which means the size of Betweenness Centrality(BC value), and locked PPARɑ as our validation targets ultimately.The nuclear hormone receptor (NR) transcription factor PPAR belongs to the family of PPARs, which are fatty acid-activated transcription factors[43].As a major regulator of substrate delivery and substrate oxidation, PPAR mainly affects genes involved in oxidative phosphorylation (OXPHOS)[44, 45].To be specific,we demonstrated that Eri exerted its anti-AML effects by activating PPARɑ protein expression but not mRNA expression through Western blot. We then used the PPARɑ inhibitor GW6471 and found that the anti-proliferation, pro-apoptotic effect of Eri on AML cells were neutralized to some extent .These results strongly confirmed that PPARɑ is the crutial target of Eri on AML cells.

Next, we wonder the specific pathways and molecular mechanisms involved in Eri acting through PPARɑ.It is reported that activated PPARs up-regulate expression of PTEN and PTEN negatively regulated PI3K/AKT pathway by converting PIP3 to PIP2[46].The PI3K/Akt pathway is abnormally activated in 60–80% of acute myelogenous leukemia (AML) patients. Given its crucial role in cell growth, proliferation, and survival, targeting this pathway for inhibition has been proposed [47]. Similarly,when we comprehensively ranked potential targets of Eri based on MCC values,PIK3R1 caught our attention since it was the predominant regulatory form of PI3K.Specifically, the PIK3R1 gene encodes the p85 and p55 and p50 splicing variants of the p85 regulatory subunit. It is reported that the abnormal expression in PIK3R1/P85ɑ could activate the PI3K/Akt pathway as well as promoting cell growth, survival and proliferation[48].More importantly,according to the results of KEGG and GO enrichment pathways,Eri was also found to be enriched in PI3K/Akt pathway. So we made a bold hypothesis that Eri could regulate the expression of PIK3R1 by activating PPARα, thereby controlling the PI3K/Akt pathway to extert anti-tumor efffect. According to the results of the Western blot, we firstly illustrated that Eri significantly inhibited the expression of PI3K/ AKT-related pathways.Additionally ,the inhibitory effect of Eri on the PI3K/Akt pathway was also reversed to some extent by GW6471.To further confirm the impact of Eri on the PI3K/AKT pathway, we used LY294002 ,the ihibitor of PI3K pathway,and found that the combination with Eri contributed to synergistic killability to AML cells. Above evidences suggest that Eri is mainly acts anti-AML effect by inhibiting the PI3K/Akt pathway through PPARɑ.The results of molecular docking also verified that Eri could combined PPARα and PIK3R1 with high affinity, which also further verified that they were the major targets of Eri in AML.As expected, we found that Eri could reduce the expression of PIK3R1 at the protein as well as the transcriptional levels,and this phenomenon could be restored by GW6471, a PPARα inhibitor.Above results provide detailed information on the molecular mechanisms by which Eri induces mitochondrial apoptosis in AML cells.

In summary, we demonstrate that Eri is a promising natural anti-cancer drug and show a vigoroso ability of pro-apoptotic and anti-proliferative on AML cells.In terms of mechanism,Eri inhibits the mRNA level of PI3KR1 gene mainly by activating the expression of PPARɑ, and ultimately inhibits the PI3K/Akt pathway.Additionally, our study provides a promising strategy that combines Eri with LY294002 and may be a more effective alternative to AML treatment.

In conclusion,we demonstrates that Erianin can exert anti-AML possibly through PPARɑand down-regulate PI3K/Akt pathways. Notably,we also preliminarily verify that the combined action

of Erianin and LY294002 can restrain the proliferation of AML cells synergistically.To summarize, our study proposes a prospective treatment strategy that the potential of Erianin to become an effective AML therapeutic strategy.In the other side,our results replenish the current perception in regard to the mechanisms of Erianin anti-AML effect,which also lays the foundation for future investigation directions, especially like the definite mechanism by which Erianin activates PPARɑ, and the reasonable exploration of effective concentration and solubility of Eri for the furthur clinical treatment.

Competing Interests

The authors proclaim hat they are not involved with or affiliated with any organization or entity that has any financial or non-financial interest in this article.

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

As an observational study,the Medical Ethical Committee of Yongchuan Hospital of Chongqing Medical University has confirmed that the ethical approval is unnecessary.

Funding

This study got financial supports by grants from the Graduate Scientific Research Innovation Project of Chongqing (CYS23349),the Key Technology Innovation Special of Key Industries of the Chongqing Science and Technology Bureau (Grant Number:csct2022ycjh-bgzxm0034).Funding sources aim to provide financial support for this research and have not otherwise involvement in this research.

Author Contribution

All authors participate in the conception and design of this research.The original draft of the manuscript,including methodology,formal analysis and investigation, was written by Ying Deng.Conceptualization, project supervision were performed by Liang Zhong. Yi Zhao, Peng Wan, Ying Zhang,Yang Liao and HongYan Zhang made a contribution to the review and editing.The final version of the manuscript was approved and accepted by all authors.

Data Availability

All data supporting the research are available from the indicated corresponding author .

Newell LF, Cook RJ (2021) Advances in acute myeloid leukemia.BMJ (Clinical research ed.), 375, n2026. https://doi.org/10.1136/bmj.n2026
Kantarjian HM, Kadia TM, DiNardo CD, Welch MA, Ravandi F (2021) Acute myeloid leukemia: Treatment and research outlook for 2021 and the MD Anderson approach.Cancer,127. 81186–1207. https://doi.org/10.1002/cncr.33477
[4]Buyel JF (2018) Plants as sources of natural and recombinant anti-cancer agents.Biotechnology advances,36. 2506–520. https://doi.org/10.1016/j.biotechadv.2018.02.002
Farhan M (2022) Green Tea Catechins: Nature's Way of Preventing and Treating Cancer. Int J Mol Sci 23(18):10713. https://doi.org/10.3390/ijms231810713
Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70(3):461–477. https://doi.org/10.1021/np068054v
Yang Z, Liu R, Qiu M, Mei H, Hao J, Song T, Zhao K, Zou D, Wang H, Gao M (2023) The roles of ERIANIN in tumor and innate immunity and its' perspectives in immunotherapy.Frontiers in immunology,14. 1170754. https://doi.org/10.3389/fimmu.2023.1170754
Zhang X, Hu L, Xu S, Ye C, Chen A (2021) Erianin: A Direct NLRP3 Inhibitor With Remarkable Anti-Inflammatory Activity.Frontiers in immunology,12. 739953. https://doi.org/10.3389/fimmu.2021.739953
Su C, Zhang P, Liu J, Cao Y, Biomedicine (2017) pharmacotherapy = Biomedecine pharmacotherapie, 88, 521–528. https://doi.org/10.1016/j.biopha.2017.01.090
Ouyang P, He X, Yuan ZW, Yin ZQ, Fu H, Lin J, He C, Liang X, Lv C, Shu G, Yuan ZX, Song X, Li L, Yin L (2018) Erianin against Staphylococcus aureus Infection via. Inhibiting Sortase Toxins 10(10):385. https://doi.org/10.3390/toxins10100385
Meng X, Yu X, Liu C, Wang Y, Song F, Huan C, Huo W, Zhang S, Li Z, Zhang J, Zhang W, Yu J (2019) Effect of ingredients from Chinese herbs on enterovirus D68 production.Phytotherapy research: PTR. 33(1):174–186. https://doi.org/10.1002/ptr.6214
Wei X, Liu J, Xu Z, Wang D, Zhu Q, Chen Q, Xu W (2024) Research progress on the pharmacological mechanism, in vivo metabolism and structural modification of Erianin. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie,173, 116295 https://doi.org/10.1016/j.biopha.2024.116295
Wang Y, Chu F, Lin J, Li Y, Johnson N, Zhang J, Gai C, Su Z, Cheng H, Wang L, Ding X (2021) Erianin, the main active ingredient of Dendrobium chrysotoxum Lindl, inhibits precancerous lesions of gastric cancer (PLGC) through suppression of the HRAS-PI3K-AKT signaling pathway as revealed by network pharmacology and in vitro experimental verification. J Ethnopharmacol 279:114399. https://doi.org/10.1016/j.jep.2021.114399
Luo Q, Li X, Gan G, Yang M, Chen X, Chen F (2021) PPT1 Reduction Contributes to Erianin-Induced Growth Inhibition in Oral Squamous Carcinoma Cells. Front cell Dev biology 9:764263. https://doi.org/10.3389/fcell.2021.764263
Chen, P., Wu, Q., Feng, J., Yan, L., Sun, Y., Liu, S., Xiang, Y., Zhang, M., Pan,T., Chen, X., Duan, T., Zhai, L., Zhai, B., Wang, W., Zhang, R., Chen, B., Han, X.,Li, Y., Chen, L., Liu, Y., … Sui, X. (2020). Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis. Signal transduction and targeted therapy, 5(1), 51. https://doi.org/10.1038/s41392-020-0149-3
Wang, P., Jia, X., Lu, B., Huang, H., Liu, J., Liu, X., Wu, Q., Hu, Y., Li, P., Wei,H., Liu, T., Zhao, D., Zhang, L., Tian, X., Jiang, Y., Qiao, Y., Nie, W., Ma, X.,Bai, R., Peng, C., … Liu, K. (2023). Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2. Signal transduction and targeted therapy, 8(1), 96. https://doi.org/10.1038/s41392-023-01329-3
Li YM, Wang HY, Liu GQ (2001) Erianin induces apoptosis in human leukemia HL-60 cells. Acta Pharmacol Sin 22(11):1018–1022
Vamecq J, Latruffe N (1999) Medical significance of peroxisome proliferator-activated receptors.Lancet(London,England),354. 9173141–148. https://doi.org/10.1016/S0140-6736(98)10364-1
ia G, Jiang X, Li Z, Ding X, Lei L, Xu S, Gao N (2021) Decoding the Mechanism of Shen Qi Sha Bai Decoction in Treating Acute Myeloid Leukemia Based on Network Pharmacology and Molecular Docking.Frontiers in cell and developmental biology,9. 796757. https://doi.org/10.3389/fcell.2021.796757
Zhou H, Jiang Y, Huang Y, Zhong M, Qin D, Xie C, Pan G, Tan J, Deng M, Zhao H, Zhou Y, Tang Y, Lai Q, Fang Z, Luo Y, Jiang Y, Xu B, Zha J (2023) Therapeutic inhibition of PPARα-HIF1α-PGK1 signaling targets leukemia stem and progenitor cells in acute myeloid leukemia.Cancer letters. 554:215997. https://doi.org/10.1016/j.canlet.2022.215997
Xu HR, Zhang JW, Chen WL, Ning ZQ, Li XN (2019) Pharmacokinetics, Safety and Tolerability of Chiglitazar, A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Pan-Agonist, in Healthy Chinese Volunteers: A Phase I Study. Clin Drug Investig 39(6):553–563. https://doi.org/10.1007/s40261-019-00779-4
Fabiani R (2020) Antitumoral Properties of Natural Products. Molecules 25(3):650. https://doi.org/10.3390/molecules25030650
Zhang X, An Y, Cheng X (2018) Research progress of antitumor activity of erianin [J]. Anal Instrum (01), 212–216
Ming L, Zhao Y, Ma P (2022) Research progress on pharmacological activities of erianin [J].Chin. J Cell Biol 44(02):399–404
Li G, Zhang H, Lai H, Liang G, Huang J, Zhao F, Xie X, Peng C (2023) Erianin: A phytoestrogen with therapeutic potential. Front Pharmacol 14:1197056. https://doi.org/10.3389/fphar.2023.1197056
Li M, He Y, Peng C, Xie X, Hu G (2018) Erianin inhibits human cervical cancer cell through regulation of tumor protein p53 via the extracellular signal-regulated kinase signaling pathway. Oncol Lett 16(4):5006–5012. https://doi.org/10.3892/ol.2018.9267
Liu YT, Hsieh MJ, Lin JT, Chen G, Lin CC, Lo YS, Chuang YC, Hsi YT, Chen MK, Chou MC (2019) Erianin induces cell apoptosis through ERK pathway in human nasopharyngeal carcinoma. Biomed pharmacotherapy = Biomedecine pharmacotherapie 111:262–269. https://doi.org/10.1016/j.biopha.2018.12.081
Liu YT, Hsieh MJ, Lin JT, Chen G, Lin CC, Lo YS, Chuang YC, Hsi YT, Chen MK, Chou MC (2019) Erianin induces cell apoptosis through ERK pathway in human nasopharyngeal carcinoma. Biomed pharmacotherapy = Biomedecine pharmacotherapie 111:262–269. https://doi.org/10.1016/j.biopha.2018.12.081
Chen YT, Hsieh MJ, Chen PN, Weng CJ, Yang SF, Lin CW (2020) Erianin Induces Apoptosis and Autophagy in Oral Squamous Cell Carcinoma Cells. Am J Chin Med 48(1):183–200. https://doi.org/10.1142/S0192415X2050010X
Wang H, Zhang T, Sun W, Wang Z, Zuo D, Zhou Z, Li S, Xu J, Yin F, Hua Y, Cai Z (2016) Erianin induces G2/M-phase arrest, apoptosis, and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells in vitro and in vivo. Cell Death Dis 7(6):e2247. https://doi.org/10.1038/cddis.2016.138
Yang L, Hu Y, Zhou G, Chen Q, Song Z (2020) Erianin suppresses hepatocellular carcinoma cells through down-regulation of PI3K/AKT, p38 and ERK MAPK signaling pathways. Biosci Rep 40(7):BSR20193137. https://doi.org/10.1042/BSR20193137
Zhu Q, Sheng Y, Li W, Wang J, Ma Y, Du B, Tang Y (2019) Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits bladder cancer cell growth via the mitochondrial apoptosis and JNK pathways.Toxicology and applied pharmacology. 371:41–54. https://doi.org/10.1016/j.taap.2019.03.027
司旭艳王世广 &王鹏.(2020).毛兰素对急性淋巴细胞白血病Jurkat细胞体外增殖和凋亡影响.中华肿瘤防治杂志(16),1277–1283.10.16073/j.cnki.cjcpt.2020.16.01
Li YM, Wang HY, Liu GQ (2001) Erianin induces apoptosis in human leukemia HL-60 cells. Acta Pharmacol Sin 22(11):1018–1022
Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111. https://doi.org/10.1038/35102167
Morgan DO (1997) Cyclin-dependent kinases: engines, clocks, and microprocessors.Annual. Rev cell Dev biology 13:261–291. https://doi.org/10.1146/annurev.cellbio.13.1.261
King KL, Cidlowski JA (1998) Cell cycle regulation and apoptosis.Annual. Rev Physiol 60:601–617. https://doi.org/10.1146/annurev.physiol.60.1.601
Xu Y, Fang R, Shao J, Cai Z (2021) Erianin induces triple-negative breast cancer cells apoptosis by activating PI3K/Akt pathway.Bioscience reports,41. 6BSR20210093. https://doi.org/10.1042/BSR20210093
Levine AJ, Oren M (2009) The first 30 years of p53: growing ever more complex.Nature reviews. Cancer 9(10):749–758. https://doi.org/10.1038/nrc2723
Liu Y, Su Z, Tavana O, Gu W (2024) Understanding the complexity of p53 in a new era of tumor suppression.Cancer cell,42. 6946–967. https://doi.org/10.1016/j.ccell.2024.04.009
Porter AG, Jänicke RU (1999) Emerging roles of caspase-3 in apoptosis.Cell death and differentiation,6. 299–104. https://doi.org/10.1038/sj.cdd.4400476
Engeland K (2022) Cell cycle regulation: p53-p21-RB signaling.Cell death and differentiation,29. 5946–960. https://doi.org/10.1038/s41418-022-00988-z
Ashkenazi A, Fairbrother WJ, Leverson JD, Souers AJ (2017) From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors.Nature reviews. Drug discovery 16(4):273–284. https://doi.org/10.1038/nrd.2016.253
Christofides A, Konstantinidou E, Jani C, Boussiotis VA (2021) The role of peroxisome proliferator-activated receptors (PPAR) in immune responses.Metabolism: clinical and experimental,114. 154338. https://doi.org/10.1016/j.metabol.2020.154338
Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ (2001) Nuclear receptors and lipid physiology: opening the X-files. Sci (New York N Y) 294(5548):1866–1870. https://doi.org/10.1126/science.294.5548.1866
Berger J, Moller DE (2002) The mechanisms of action of PPARs. Annu Rev Med 53:409–435. https://doi.org/10.1146/annurev.med.53.082901.104018
Kitagishi Y, Matsuda S (2013) Diets involved in PPAR and PI3K/AKT/PTEN pathway may contribute to neuroprotection in a traumatic brain injury. Alzheimers Res Ther 5(5):42. https://doi.org/10.1186/alzrt208
Herschbein L, Liesveld JL (2018) Dueling for dual inhibition: Means to enhance effectiveness of PI3K/Akt/mTOR inhibitors in AML.Blood reviews,32. 3235–248. https://doi.org/10.1016/j.blre.2017.11.006
Vallejo-Díaz J, Chagoyen M, Olazabal-Morán M, González-García A, Carrera AC (2019) The Opposing Roles of PIK3R1/p85α and PIK3R2/p85β in Cancer.Trends in cancer,5. 4233–244. https://doi.org/10.1016/j.trecan.2019.02.009

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Erianin suppresses the development of acute myeloid leukemia via PPARɑ and regulating PI3K/AKT signaling pathways

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Abstract

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Materials and methods

Cell lines and culture

Chemicals and reagents

Cell proliferation and viability assays

Cellular apoptosis detection

Cell cycle analysis

Network pharmacology analysis

Molecular docking

Western Blotting

Quantitative real‑time PCR

Statistical analysis

Results

Erianin induces G2/M phase arrest in AML cells

Screening the target genes and pathways of Erianin's anti-AML activity by network pharmacology

Erianin exploits anti-AML effect through PI3K/AKT signaling pathways

Discussion

Conclusion

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Competing Interests

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