Agaricus blazei extract FA2bβ enhances the sensitivity of K562/ADR cells through the PI3K/Akt/mTOR signalling pathway

doi:10.21203/rs.3.rs-2343202/v1

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Agaricus blazei extract FA2bβ enhances the sensitivity of K562/ADR cells through the PI3K/Akt/mTOR signalling pathway

https://doi.org/10.21203/rs.3.rs-2343202/v1

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The acidic RNA protein complex FA2bβ, isolated from Agaricus blazei Murill (AbM), has proapoptotic effects and antiproliferative potency in several cancers. In the present study, we explored whether FA2bβ can boost the sensitivity of adriamycin (ADR) to multidrug-resistant chronic myelogenous leukaemia (CML) K562/ADR cells. A bioinformatics search identified differentially expressed genes (DEGs) for enrichment analyses. The synergistic antiproliferative effect of FA2bβ and ADR was investigated using a CCK8 assay. Flow cytometry was used to evaluate the cell apoptosis rate, cell cycle and autofluorescence intensity of ADR. Western blotting was used to analyse the mechanism of anti-leukaemia action. There was a synergistic effect of FA2bβ and ADR on the chemosensitivity of K562/ADR cells, showing increased apoptosis and intracellular ADR in K562/ADR cells but downregulated P-glycoprotein (P-gp) and phosphorylation of PI3K, Akt and mTOR. FA2bβ improved the sensitivity of ADR to MDR K562/ADR cells, perhaps by regulating the PI3k/Akt signalling pathway and downregulating P-gp. These results suggest that FA2bβ may be a combination therapy for CML in the future.

Chronic myelogenous leukaemia

Multidrug resistance FA2bβ

PI3K

Apoptosis

Chronic myelogenous leukaemia (CML) is a clonal haematopoietic stem cell malignancy characterized by the formation of the BCR-ABL fusion oncogene and Philadelphia chromosome (Ph)[1]. Tyrosine kinase inhibitor, such as imatinib, are preferred frontline therapies for CML and achieve remarkable treatment outcomes but require lifelong treatment[2]. Drug resistance could develop following long-term treatment for CML patients[3]. Ammar et al. [4] demonstrated that 54.3% of patients display primary or acquired resistance to IM. Multidrug resistance (MDR) is associated with the overexpression of drug transporters such as the ATP-binding cassette (ABC) protein, the activation of diverse signalling pathways, and alterations of the apoptosis pathway[5]. ABC transporters such as P-glycoprotein (P-gp) are encoded by the MDR1 gene. P-gp expression and activity have been the most studied mechanisms by which MDR occurs. There is a significant correlation between the overexpression of P-gp and the failure of the IM response in CML patients, and 3-year overall survival (OS) and progression-free survival (PFS) in CML patients with positive P-gp expression were significantly lower than those in patients with negative expression [4]. The compromised outcome is a major challenge in CML chemotherapy. Therefore, exploring novel candidates for alternative approaches with safer and more effective effects in response to the development of MDR is needed.

The phosphoinositide-3-kinase/serine-threonine kinase (PI3K/Akt) pathway is continuously activated by BCR-ABL (fusion ongene), which is involved in multiple biological processes, including cell proliferation, cell cycle, apoptosis, autophagy and drug resistance[6]. The resistant CML line K562/ADM (Adriamycin resistance) showed higher PI3K/Akt activity than sensitive K562 cells[7]. Abnormal activation of the PI3K/Akt signalling pathway plays a key role in drug resistance[8] and is closely related to the expression of MRP1 and P-gp[9]. Furthermore, inhibition of the PI3K/Akt signalling pathway can restore drug sensitivity in CML cells[10]. The combination of ponatinib (a third-generation TKI) and PI3K/mTOR dual-inhibitor therapy for CML increases the anti- leukaemia effects of ponatinib but reduces ponatinib doses with synergistic anti-leukaemia effects on leukaemia cells[11].

Natural products and traditional Chinese herbal medicine for leukaemia therapy have attracted increased attention because they have fewer adverse effects and are safe, stable and bioavailable [9, 12]. The combination of natural compounds and chemotherapy has minimal adverse effects [13]. FA2bβ is an active compound derived and isolated from AbM with many biological activities, including antiproliferation of several cancer cells by inducing apoptosis and increasing the sensitivity of anticancer drugs[14]. Our previous data demonstrate that FA2bβ inhibits proliferation but induces apoptosis of human CML K562 cells[15]. However, whether and how FA2bβ can reverse MDR remains to be explored. Thus, the aim of the present study was to investigate the effect of FA2bβ on MDR and clarify the role of the PI3K/Akt signalling pathway.

Cell Line and Culture

The human CML cell line K562 was obtained from Lanzhou University Medical School. The human CML cell line K562/ADR was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. K562 cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO₂. K562/ADR cells were cultured in medium containing 1 µg/ml ADR to maintain their MDR phenotype. However, K562/ADR cells were maintained without ADR for 2 weeks prior to experiments.

Reagents

ADR was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (cat. 2101E1). FBS was obtained from BI company (cat. no. E600001-0500). Cell Counting Kit8 (CCK-8) reagent was purchased from AbMole Company (cat. M4839-500Tests). FA2bβ was extracted from AbM, as previously described[14]. Antibodies against P-gp (cat. no. 22336-1-AP) was obtained from Proteintech, Inc., Akt (cat. no. 60203-2-Ig), mTOR (cat. no. 66888-1-Ig), β-actin (cat. no. 66009-1-Ig), BAX (cat. no. 60267-1-Ig) and Bcl-2 (cat. no. A19693) were purchased from Abcam. PI3K (cat. no. 4219), p-PI3K (cat. no. 4257), p-Akt (cat. no. 4060;), and p-mTOR (cat. no. 5536) were purchased from Cell Signaling Technology, Inc. HRP-conjugated Affinity purified goat anti-mouse IgG (cat. no. SA00001-1) or goat anti-rabbit IgG (cat. SA00001-2) were obtained from Abcam.

Rna Sequencing

We investigated the gene expression profiling between K562 and K562/ADR cells with RNA-seq, as described. (ref). Briefly, total RNA was extracted from cells using TRIzol solution. To analyse the transcriptome, RNA sequencing libraries were prepared according to the VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina (Illumina NovaSeq 6000). Genes with |log2FC|>2 and FDR < 0.5 were identified as DEGs. To identify the DEGs in K562 and K562/ADR cells, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

Cck-8 Assay

A CCK-8 assay was performed to detect cell viability. K562 and K562/ADR cells were seeded in 96-well plates (5×10⁴/well) and incubated with various concentrations of FA2bβ and/or doxorubicin for 48 h. Subsequently, the cells were further incubated with 10 µl CCK-8 solution at 37°C for an additional 4 h. The 50% inhibition of cell growth (IC50) of ADR on the two cells was calculated by GraphPad Prism software. The resistance index (RI) and the reversal fold (RF) were calculated by the following formulas:

Resistance index (RI) = IC50(K562/ADR)/IC50(K562);

Reversal fold (RF) = IC50(ADR)/IC50 (ADR + FA2bβ).

Flow Cytometry (Fcm) Analysis Of Apoptosis

Apoptosis was examined using FCM after Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. K562/ADR cells were treated with various concentrations of FA2bβ and/or doxorubicin for 24 h at 37°C. After treatment, the cells were washed three times with ice-cold phosphate buffered saline (PBS). Then, the cells were stained with 5 µl of Annexin V-FITC solution and PI solution at room temperature for 15 min in the dark. Finally, the apoptosis analysis was immediately analysed by BD flow cytometry.

Fcm Of Cell Cycle Analysis

To evaluate the effect of FA2bβ on the cell cycle, PI staining was performed using flow cytometry according to the manufacturer's instructions. Cells were seeded at 1×10⁶ cells and incubated in the absence or presence of different doses of FA2bβ for 24 h at 37°C. Cells were fixed in 70% ethanol at 4°C overnight. After washing with PBS, the cells were treated with 100 µl of RNase for 30 min at 37°C. Next, the cells were incubated with 400 µl PI at 4°C for 30 min. The stained cells were then analysed for cell cycle distribution.

Fcm For The Mean Fluorescence (Mfi) Of Adr

K562/ADR cells were cultured in a 6-well plate at a concentration of 1×10⁶/well and then treated with ADR alone or in combination with 1 mg/ml FA2bβ for 24 h. Then, 10 µM ADR was added for another 1 h at 37°C. Cells were washed three times with PBS. MFI was detected by FCM immediately.

Western Blotting (Wb) Analysis

K562/ADR cells growing in log phase were collected into a 6-well plate at a concentration of 1×10⁶ cells/well. Cells were treated with different concentrations of FA2bβ for 24 h and then harvested and washed with PBS three times. RIPA buffer was used to extract total protein at 4°C. The sample lysates were boiled, separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% fat-free milk for 1 h, the membranes were incubated with their respective primary and secondary antibodies at 4°C. Enhanced chemiluminescence (ECL) was used to measure the expression of related proteins. Finally, the results were recorded and calculated using ChemiDoc molecular imaging systems and ImageJ software. The relative protein expression was normalized to β-actin, which is widely used as an internal reference for WB.

Statistical analysis

The statistical software GraphPad Prim was used for statistical analysis of the experimental data. The experimental results are expressed as the mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant. Two groups were compared using a t test, and multiple groups were compared using one-way analysis of variance (ANOVA).

Next-generation sequencing (NGS), notably whole-transcriptome sequencing (RNA-seq)

The volcano plot shows the differential distribution of the expression levels of genes between K562 and K562/ADR cells. A total of 1415 DEGs were identified, including 564 upregulated genes and 851 downregulated genes (Fig. 1A).

GO analysis results showed that changes in the molecular function (MF) of DEGs were significantly enriched in sequence-specific double-stranded DNA binding and signalling receptor activity. Changes in biological process (BP) were mainly enriched in signal transduction. Changes in the cell component (CC) of DEGs were mainly enriched in the plasma membrane, integral component of membrane, and extracellular vesicular exosome (Fig. 1B). The results of KEGG pathway analysis showed that DEGs were mainly enriched in the PI3K/Akt signalling pathway, ABC transporter, and mitophagy-animal were significantly increased in K562/ADR cells compared to K562 cells (P < 0.01) (Fig. 1C).

Multidrug Resistance Of K562/adr Cells

To verify the drug-resistance characteristics of K562/ADR cells, the cytotoxicity of ADR was measured in K562/ADR and K562 cells by CCK-8 assay. The IC50 values of ADR on K562 and K562/ADR cells were 0.75 µM and 20.34 µM, respectively. The RI was calculated as 26.88 (P < 0.05). Cell proliferation testing showed that compared with K562 cells, K562/ADR cells showed clear drug resistance to chemotherapeutic drugs (Fig. 2).

The Expression Of P-gp In K562 And K562/adr Cells

We also explored the expression of P-gp in K562 and K562/ADR cells by WB. We found that the protein levels of P-gp in K562/ADR cells were notably increased and were higher than those in K562 cells (Fig. 3).

Anti-proliferative Activity Of Fa2bβ On K562 And K562/adr Cells

We then investigated the anti-proliferative effects of FA2bβ on K562 and K562/ADR cells. The cells of both cell lines were treated with 0.5,1,1.5,2,2.5 and 3 mg/ml concentrations of FA2bβ for 48 h, and the cell viability was analysed with CCK8 assay. As shown in Fig. 4, FA2bβ inhibited proliferation of both cell lines in a dose-dependent manner, the IC50 values of K562 and K562/ADR cells were 1.48 mg/ml and 1.62mg/ml, respectively.

Fa2bβ Induced Reversal Of The Resistance Of K562/adr Cells To Adr

The antiproliferative effect of FA2bβ on K562/ADR cells was investigated using a CCK8 assay. The IC50 values of ADR against K562/ADR cells were 20.16 ± 0.77 µM, 14.94 ± 0.51 µM, and 12.10 ± 0.79 µM for ADR plus 0.5 mg and 1 mg FA2bβ, respectively. The RF change of 0.5 and 1 mg FA2bβ was 1.35 and 1.67, respectively. Combination treatment resulted in a synergistic increase in the sensitivity of K562/ADR cells to ADR (Fig. 5). The reversal effect of FA2bβ on drug resistance in K562/ADR cells was dose dependent.

Effect Of Fa2bβ On The Intracellular Accumulation Of Adr

The MFI of ADR was measured by FCM and was significantly decreased in K562/ADR cells compared with K562 cells. However, the intracellular accumulation of ADR gradually increased when K562/ADR cells were treated with FA2bβ and ADR, demonstrating that in combination with FA2bβ, K562/ADR cells exhibited a higher level of intracellular ADR (Fig. 6).

Fa2bβ Enhanced Apoptosis In K562/adr Cells

Our previous studies reported that FA2bβ could induce apoptosis of K562 cells. To further investigate the combined effect of FA2bβ and ADR, K562/ADR cells were incubated with FA2bβ and ADR for 24 h.

As shown in Fig. 7, 1 mg FA2bβ or 10 µM ADR slightly increased the apoptotic proportions of K562/ADR cells, with apoptotic rates of 20.95 ± 2.65% and 14.28 ± 0.66%, respectively. In contrast, the combination of 1 mg FA2bβ showed significant apoptosis, with an apoptotic proportion of 39.23 ± 3.81%. These results showed that the combination of FA2bβ and ADR significantly increased the apoptotic rate of K562/ADR cells in a dose-dependent manner. Based on the flow cytometry results, the combination of the two drugs dramatically suppressed growth and increased the percentage of cells in early apoptosis and late apoptosis.

Fa2bβ Induced Cell Cycle Arrest In K562/adr Cells

To further investigate whether FA2bβ can influence the growth of K562/ADR cells through cell cycle distribution, we detected the cell cycle distribution after FA2bβ and/or doxorubicin exposure. The cell cycle distribution underwent significant changes in all treatment groups when compared with the control group. FA2bβ plus ADR induced cell cycle arrest in the G0/G1 phase compared with the ADR treatment group. Increasing the FA2bβ concentration increased the proportion of cells in the G1 phase and gradually decreased the proportion of cells in the S phase. These results indicated that FA2bβ stimulation could change the cell cycle distribution by inducing cell cycle arrest at the G1 phase (Fig. 8).

Fa2bβ Decreases The Expression Of P-gp In K562/adr Cells Via The Pi3k/akt Signalling Pathway

To explore the mechanism and the role of the PI3K/Akt signalling pathway in the FA2bβ-mediated reversal of multidrug resistance, we analysed the expression of P-gp, Bax, Bcl-2 and PI3k/Akt signalling pathway proteins in K562/ADR cells. The cells were treated with FA2bβ alone and combined with ADR for 24 h. Western blot results showed that the combination of FA2bβ and ADR significantly decreased the expression of P-gp, PI3K, p-PI3K, Akt, p-Akt, and mTOR. However, the expression of the corresponding total proteins showed no obvious changes. Altogether, this study suggested that FA2bβ combined with ADR might inhibit the proliferation and cell cycle progression of K562/ADR cells via the PI3K/Akt/mTOR signalling pathway. Meanwhile, the result showed that the combination treatment of K562/ADR cells with FA2bβ and ADR resulted in a marked increase in Bax expression, and a significantly downregulation of Bcl-2 (Fig. 9).

In the present study, we showed that K562/ADR cells were more resistant to the chemotherapeutic drug ADR by a high RI value and that the protein levels of P-gp in K562/ADR cells were notably increased. RNA-seq results showed that the PI3K/Akt signalling pathway, ABC transporter, and autophagy were significantly increased in K562/ADR cells compared to K562 cells. Combination treatment with ADR and FA2bβ in K562/ADR cells resulted in a synergistic increase in the sensitivity of K562/ADR cells to ADR. In addition, the combined effect of FA2bβ enhanced apoptosis in K562/ADR cells, induced cell cycle arrest, and decreased the expression of P-gp in K562/ADR cells via the PI3K/Akt signalling pathway.

MDR is one of the most notable problems in leukaemia chemotherapy, and approximately 40% of all CML patients fail TKI treatment in the presence of sustained BCR-ABL1 inhibition[16]. During the disease course, 20–30% of patients display drug resistance[17]. Patients with positive expression of P-gp are less likely to achieve a major molecular response (MMR) and have a higher risk of resistance to IM[4]. Therefore, it is very important to reverse the MDR of CML and improve the prognosis and survival rate of patients. At present, a combination of several drugs simultaneously reduces the side effects of drugs, reduces the dose of antitumour drugs and improves the efficacy[18]. Combined IM and ATRA therapy for CML was effective in decreasing ABCB1 and BCR-ABL genes, demonstrating a new therapeutic option for those patients who develop resistance to available treatments[19]. Matrine combination treatment with doxorubicin enhanced apoptosis induced by DOX in K562 and K562/ADR cells[20].

We found that after cotreatment with FA2bβ and ADR, the IC50 values of ADR declined. Our results demonstrated that FA2bβ at a lower dose significantly enhanced ADR-induced antiproliferative activity against K562/ADR cells, indicating that FA2bβ might affect ADR resistance in K562/ADR cells.

Altered apoptosis resistance was also described as a mechanism of MDR[21], and the combination of FA2bβ and ADR significantly enhanced ADR-induced apoptosis in CML cells based on flow cytometry, accompanied by an increase in the percentage of early apoptosis and late apoptosis. Furthermore, FA2bβ-induced apoptosis in K562/ADR cells was initiated by the pro- and antiapoptotic proteins Bax/Bcl-2, showing that the cytotoxicity of FA2bβ towards K562/ADR cells was due to reduced apoptosis resistance. Our data suggested that FA2bβ and ADR combined treatment sensitized K562/ADR cells and induced apoptosis, consistent with reports of FA2bβ-induced apoptosis in K562 cells.

A previous study revealed that FA2bβ can arrest the cell cycle at the G1 phase in K562 cells[15]. Cell cycle progression seems to be a common mechanism for drug-resistance in CML[22]. In the current study, our results demonstrated that G1 phase arrest was induced by FA2bβ treatment in K562/ADR drug‑resistant cell lines, and cell cycle arrest in G0/G1 phase was increased from 39.44–58.66%, resulting in G0/G1 phase cell cycle arrest.

K562/ADR cells express high levels of MDR1 and show resistance not only to ADR but also to a large range of anticancer reagents, including etoposide, vincristine, and IM[23]. In addition, P-gp acts as a drug pump and can mediate the efflux of various intracellular chemotherapeutic drugs, resulting in insufficient therapeutic concentrations and the development of IM resistance, such as methotrexate, vincristine and ADR[24]. The reduction in intracellular drugs is one cause of drug resistance in K562/ADR cells. Our results indicated that FA2bβ may elevate sensitivity and reverse drug resistance by increasing intracellular ADR accumulation, suggesting that this is the mechanism by which FA2bβ reverses MDR.

The BCR-ABL fusion oncogene can encode the BCR-ABL protein, which activates multiple downstream signalling pathways, including the PI3K/Akt, MAPK, and RAS pathways[25]. Aberrant activation of the PI3K/Akt signalling pathway is widely observed in blood cancer and other malignant tumours. Additionally, constitutive activation of the PI3K/Akt signalling pathway plays an important role in the pathogenesis of MDR and is associated with poor prognosis[26]. There is a close relationship between the activity of the PI3K/Akt signalling pathway and the levels of P-gp [7]. Additionally, our results suggested that cells treated with FA2bβ combined with ADR exhibited significantly downregulated expression of p-PI3K, p-Akt, and p-mTOR in K562/ADR cells, accompanied by inactivation of the protein levels of the multidrug resistance protein P-gp. The results demonstrated that the reduced expression of p-PI3K and p-Akt could downregulate the activity of the PI3K/Akt signalling pathway.

Therefore, our findings suggest that FA2bβ could be used as a therapeutic adjunct in CML therapy to enhance the efficacy of chemotherapy drugs. The combined therapy of a low dose of FA2bβ and ADR showed high efficacy and significantly reduced the degree for cases of resistant cells. There were limitations in this study. Whether FA2bβ could reverse drug resistance through other signalling pathways was not fully analysed in this research, and this limitation might be gradually improved in future studies.

In summary, FA2bβ reversal of MDR was associated with FA2bβ potentiating the sensitivity of K562/ADR cells to ADR, increasing the intracellular accumulation of ADR, and FA2bβ and ADR synergistically enhancing the induction of apoptosis and significantly suppressing the protein expression of the resistance-related protein P-gp. Moreover, the Bcl-2/BAX and PI3K/Akt signalling pathways were inhibited. These findings indicated that the application of FA2bβ might be a new MDR reversal agent in the treatment of CML.

Acknowledgements

We thank teacher chao xu from the first affiliated hospital of Soochow university for providing us with the technical guidance.

Conflict of interest

We declare that we have no conflict of interest.

Author Contributions

S.Y.Q and W.D.P designed the study; G.W.W and W.D.P performed the experiments, analysed data and wrote the article; S.Y.Q revised the manuscript. All authors have read and approved the final manuscript.

Funding information

This work was supported by the National Natural Science Foundation of China (Nos. 81560670), the Natural Science Foundation of Gansu Province (grant no. 20JR10RA376 and 21JR11RA196), Science and Technology Planning project from Lanzhou city, Lanzhou, Gansu Province, China (grant no.2020-ZD-56)

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Agaricus blazei extract FA2bβ enhances the sensitivity of K562/ADR cells through the PI3K/Akt/mTOR signalling pathway

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Abstract

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Introduction

Materials And Methods

Cell Line and Culture

Reagents

Rna Sequencing

Cck-8 Assay

Flow Cytometry (Fcm) Analysis Of Apoptosis

Fcm Of Cell Cycle Analysis

Fcm For The Mean Fluorescence (Mfi) Of Adr

Western Blotting (Wb) Analysis

Statistical analysis

Results

Next-generation sequencing (NGS), notably whole-transcriptome sequencing (RNA-seq)

Multidrug Resistance Of K562/adr Cells

The Expression Of P-gp In K562 And K562/adr Cells

Anti-proliferative Activity Of Fa2bβ On K562 And K562/adr Cells

Fa2bβ Induced Reversal Of The Resistance Of K562/adr Cells To Adr

Effect Of Fa2bβ On The Intracellular Accumulation Of Adr

Fa2bβ Enhanced Apoptosis In K562/adr Cells

Fa2bβ Induced Cell Cycle Arrest In K562/adr Cells

Fa2bβ Decreases The Expression Of P-gp In K562/adr Cells Via The Pi3k/akt Signalling Pathway

Discussion

Conclusion

Declarations

References

Status:

Version 1