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. 2022 Oct 26;39(2):208–219. doi: 10.1007/s12288-022-01573-7

Synergistic Effects of PI3K Inhibition on Arsenic Trioxide Cytotoxicity in Acute Promyelocytic Leukemia Cells: A New Portrait of Idelalisib as an Adjuvant Therapy

Maryam Dadashi 1, Atieh Pourbagheri-Sigaroodi 1, Ali Anjam-Najmedini 1, Davood Bashash 1,✉
PMCID: PMC10064366  PMID: 37006983

Abstract

The advent of small-molecule inhibitors targeting the components of oncogenic signaling pathways has revolutionized cancer treatment, where the pharmacological approaches have gone from an era of non-specific chemotherapeutic drugs to the golden age of targeted therapies. In the present study, we evaluated the therapeutic value of an isoform-specific inhibitor of PI3K (Idelalisib) in potentiating the anti-leukemic effects of arsenic trioxide (ATO), an eminent drug used in the treatment of acute promyelocytic leukemia (APL). We found that the abrogation of the PI3K axis profoundly reinforced the anti-leukemic effects of the lower concentrations of ATO, as revealed by the superior reduction in the viability, cell number, and metabolic activity of APL-derived NB4 cells as compared to either agent alone. The cytotoxic effect of Idelalisib in combination with ATO was probably mediated through suppression of c-Myc that was coupled with the elevation in the intracellular level of reactive oxygen species and induction of caspase-3-dependent apoptosis. Notably, our results showed that the suppression of autophagy reinforced the ability of the drugs in eradicating the leukemic cells, suggesting that the compensatory activation of this system may probably overshadow the success of Idelalisib-plus-ATO in APL cells. All in all and given the significant efficacy of Idelalisib against NB4 cells, we proposed the application of this PI3K inhibitor as a foreseeable approach with a safe profile in the treatment of APL.

Keywords: acute promyelocytic leukemia, PI3K inhibition, Idelalisib, ATO, Autophagy

ORCID ID: 000-0002-8029-4920.

Ethical statements

This material is the authors’ own original work, which has not been previously published elsewhere and the paper reflects the authors’ own research and analysis in a truthful manner. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors and informed consent is not required. This study is approved by the ethical committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1396.969).

Synergistic effects of PI3K inhibition on arsenic trioxide cytotoxicity in acute promyelocytic leukemia cells: a new portrait of Idelalisib as an adjuvant therapy.

Introduction

Since the last years of the twenty century, when the results of the deep molecular and genetic experiments began to accuse the aberrant activation of signaling pathways in the evolution of human cancers, therapeutic approaches for cancer patients did not change for many years. The number of patients who underwent disease relapse increased day by day and the application of intensive chemotherapy regimens and high doses of radiation were the only approaches used for battling the demon of cancer, which itself confront patients with devastating side effects [1]. It became apparent the propagation of oncogenic signaling pathways endow neoplastic cells with a survival advantage through blunting the apoptotic stimuli induced by chemotherapeutic drugs or radiotherapy [2]. Like any type of other human cancer, acute promyelocytic leukemia (APL), known as AML-M3, benefits from the activated cell signaling pathways, which provide them a fertile ground for maintaining their viability and proliferative capacity [3]. PI3K/Akt signaling pathway is one of the main networks that is activated in most cases of this type of leukemia, which not only has a hand in the disease progression [4] but also has a fundamental role in the induction of chemoresistance [5]. Several reports have reported that the activation of the PI3K/Akt signaling axis could compensatorily bypass the effectiveness of a wide range of chemotherapeutic drugs in APL-derived leukemic cell lines, in particular ATO [6, 7], a novel drug that changed the conventional landscape of APL treatment [8]. Currently, the concentration of 0.15 mg kg− 1 body weight/day is suggested for the clinical administration of ATO in APL patients, which is equivalent to 1–2 µM in in vitro experiments [9].

Moreover, a large body of evidence proved the superior effects of all-trans retinoic acid (ATRA), the first-line drug of choice for the treatment of patients with new-diagnosed, in the presence of PI3K inhibitors. For instance, Billottet et al. reported that ATRA led to the activation of PI3K and improved cell survival in the presence of cytotoxic agents such as doxorubicin, and the addition of class I PI3K inhibitors could significantly improve the antiproliferative effects of ATRA in both NB4 and primary APL cells [10]. In another study, Swords et al. showed that pro-differentiation effects of ATRA are markedly potentiated when combined with PI3K/AKT/mTOR inhibitor, and loss of cell viability, cell cycle arrest in the G1 phase, and impaired survival were more prominent than with any agent used alone [11].

Although the effects of different types of PI3K inhibitors in different human cancers have been investigated [12], the identification of detailed mechanisms of action by which these inhibitors reduce the viability of cancer cells is still challenging. Idelalisib (CAL-101), an isoform-specific PI3K inhibitor, displays linear pharmacokinetics with favorable cytotoxic ability in different types of human cancers [13]. Idelalisib has received its FDA approval in 2014 for the treatment of B cell hematological cancers, in particular in relapsed patients suffering from chronic lymphocytic leukemia (CLL), follicular B-cell non-Hodgkin lymphoma (NHL), and small lymphocytic leukemia (SLL) [14]. The success of this agent in endowing the survival to refractory cancer patients paved the way forclinical application of Idelalisib and multiple studies proved that the anti-tumor activities of this PI3K inhibitor are not limited to lymphocyte-derived leukemia and there are several reports about its anti-tumor efficacy in human leukemia originated from myeloid lineage. In a study conducted by Nguyen et al., the therapeutic value of Idelalisib on AML patients samples and cell lines has been examined and their results showed that this agent could robustly halt cell proliferation through suppression of ribosomal RNA synthesis [15]. It has also been shown that by reducing p-Akt level, inhibition of other PI3K axis effectors, such as -p70S6K, p-GSKb, and p-Bad, Idelalisib could exert favorable anti-leukemic effects in acute myeloid leukemia cells [16]. The results of the Phase I clinical trial in patients with relapsed acute myeloid leukemia put Idelalisib at the center of attention as a foreseeable approach with a safe profile in the treatment of AML [17]. The results of the clinical pharmacokinetics of Idelalisib revealed that this drug is well-tolerated at the dose of 400 mg/day, corresponding to a maximum concentration of about 20 µM in pre-clinical investigations [18]. Once there is a discussion about the therapeutic efficacy of an agent, many variables enter into the equation and there would be a long way to determine the proper behavior of the agent in the presence of other anti-cancer agents. In an effort to explore the potential therapeutic value of PI3K inhibitor as an adjuvant approach in APL treatment, we designed experiments to assess the synergistic effect between Idelalisib and the lower concentrations of ATO in APL-derived NB4 cells.

Materials and methods

Cell culture and drug treatment

To investigate the effects of Idelalisib and ATO in acute promyelocytic leukemia cells, NB4 cells were cultured in RPMI 1640 medium supplemented with antibiotics, 10% fetal bovine serum (Invitrogen) and 2 mM l-glutamine (Invitrogen) in the humidified incubator. Cells were treated with ATO (Sina Daroo, Iran) and Idelalisib (Selleckchem, Germany), either as single agents or in a combined modality. For further analysis, cells were also exposed to an inhibitor of autophagy chloroquine (Sigma Germany). The cells were also exposed to equal amounts of solvents, as an alternative control at the final concentration of 0.1%.

Trypan blue assay test of cell count and viability

The viability and the proliferative capacity of NB4 cells in response to Idelalisib, ATO, CQ, and the combination treatment were evaluated by using Trypan blue assay. Cells were exposed to the relevant concentrations of the agents and after the indicated time intervals, the drug-treated cells were collected and mixed with a 0.4% trypan blue solution (Invitrogen) in a 1:1 ratio. After incubation for 1–2 min at room temperature, each sample was loaded onto the chamber of the Neubauer hemocytometer and the proportion of viable cells was calculated by dividing the number of non-stained (viable) cells by the total number of cells.

Detection of metabolic activity by microculture tetrazolium test

NB4 cells at the concentration of 5 × 103 were cultured into 96-well plates and then were treated with Idelalisib, ATO, CQ, either as single agents or in a combined modality up to 36 h. To assess whether the aforementioned agents could reduce the metabolic activity of the cells, 100 µl of MTT solution was added to each well at the end of the drug incubation time. After 3 h of incubation at 37 οC, the plate was centrifuged and the discarded media of each well was replaced by 100 µl of sterile DMSO. The optical densitometry of dissolved formazan was measured in an enzyme-linked immunosorbent assay (ELISA) reader at the wavelength of 570 nm. The percentage of metabolic activity was calculated as (%) = (ODexp/ ODcon) ×100; where ODexp and ODcon are the optical densities of exposed and control cells, respectively.

BrdU proliferation assay

To evaluate the effect of Idelalisib and ATO on the proliferative capacity of NB4 cells, the DNA synthesis rate of the cells was measured using the BrdU cell proliferation kit (Roche, Basel, Switzerland) and according to the manufacturer’s instruction. Briefly, after exposure of NB4 cells to 10 µM of Idelalisib and 1 µM of ATO either as a single agent or in a combined modality for 24 and 36 h, cells were incubated with 100 µl/well of BrdU labeling solution. The cells were fixed, DNA was denatured using 200 µl of Fixodent solution and then a peroxidase-conjugated mouse monoclonal anti-BrdU-POD antibody was added. Immune complex formation was measured at 450 nm using an ELISA Reader. Absorbance values were directly correlated with the amount of DNA synthesis and thereby with the number of proliferating cells.

Determination of combination index (CI) and dose reduction index (DRI)

To investigate the efficacy of drug combinations, the reduction of cell survival was examined and the combination index (CI) and dose reduction index (DRI) were evaluated as described previously [19]. The CI values of < 1, =1, and > 1 indicate synergism, additive effect, and antagonism of drugs, respectively.

Cell cycle distribution analysis

The influence of Idelalisib and ATO on the progression of the cell cycle was evaluated using flow cytometry. After 24 h of drug treatment, 1 × 106 NB4 cells were collected, washed twice with cold PBS, and then fixed in 70% ethanol. To stain DNA and degrade RNA, propidium iodide (PI) and RNase were added to each sample. The proportion of the cells in each phase of the cell cycle was quantified from flow cytometric histograms. For interpretation of the results, we used the Windows FlowJo V10 software.

Assessment of apoptosis using flow cytometry

NB4 cells were cultured in 24-well plates and were exposed to Idelalisib (10µM), ATO (1 µM), and the combination of the two drugs for 24 h. The harvested cells were washed with PBS and 100 µl of the incubation buffer were added to each sample. Cells were then suspended and incubated for 20 min with Annexin-V-Flous solution in dark. The percentage of annexin-V and annexin-V/PI positive cells was quantified by using flow cytometry and the Windows FlowJo V10 software.

Measurement of caspase-3 enzymatic activity

To determine whether the induction of apoptosis was mediated through activation of caspase-3 activity, we examined the enzymatic activity of caspase-3 using a caspase-3 assay kit (Sigma). After 24 h incubation of NB4 cells to the identical drug concentration that was used for the annexin-V/PI staining assay, cells were centrifuged at 600 × g for 5 min, and then they were lysed. The lysates underwent one more stage of centrifugation and 20,000 × g for 10 min and then 5 µg of the supernatant was incubated with 85 µl of assay buffer plus 10 µl of the caspase-3 substrate in a 96-well plate at 37 °C. Cleavage of the peptide by caspase-3 released the chromophore pNA, which was quantified spectrophotometrically at 405 nm.

RNA extraction, cDNA synthesis, and quantitative real-time PCR

Total RNA from APL-derived NB4 cells was extracted using the RNA Isolation Kit (Roche, Mannheim, Germany). After confirming the quantity of the extracted RNA by Nanodrop instrument, the reverse transcription reaction was performed using the cDNA synthesis kit (Takara Bio, Shiga, Japan) to synthesize the relevant cDNAs of each extracted RNA. Next, to evaluate the effect of Idelalisib/ATO on the expression of proliferation- and apoptotic-related genes, the thawed cDNAs were subjected to quantitative real-time PCR (qRT-PCR). The fold change values were calculated based on the 2−ΔΔCt relative expression formula.

Intracellular reactive oxygen species detection

To determine the effect of Idelalisib, ATO, and Idelalisib-plus-ATO on the amount of intracellular reactive oxygen species (ROS) in NB4 cells, we incubated the drugs-treated cells with a fluorogenic dye DCFH-DA for 30 min. The fluorescence intensities of the samples were quantified by a fluorescence spectrophotometer (Cary Eclipse, USA) with excitation at 485 nm and emission at 530 nm.

Detection of autophagy by acridine orange staining

To assess the effect of autophagy inhibition on the cell sensitivity to the agents, NB4 cells were treated with an increasing concentration of CQ for 24 h. Then, the relevant amounts of cells were harvested and washed three times with PBS. 1 µg/mL acridine orange stain (Merck, Germany) was then added to each sample and the cells were incubated in the dark for the next 15 min. The difference in the autophagic lysosomes was visualized by a fluorescence microscope (Labomed, Los Angeles). The autophagic lysosomes have different acidity as compared to cytoplasm and nucleus. For this reason, in contrast to cytoplasm and nucleus are stained as green, these lysosomes appear as orange/red fluorescent cytoplasmic vesicles.

Statistical analysis

Experimental data were evaluated in triplicate against untreated control cells and collected from three independent experiments. All presented data were analyzed using GraphPad Prism Software using a two-tailed student’s test and one-way variance analysis. In order to compare between the control group and the drugs-treated ones, Dennett’s multiple comparison test was used. All data are presented as mean ± standard deviation (S.D.) and a probability level of P ≤ 0.05 was considered statistically significant.

Results

Idelalisib promoted ATO-induced cytotoxic effect in APL- derived NB4 cells

Activation of the PI3K pathway not only contributes to numerous cellular processes including cell survival and growth but also results in drug resistance in cancer cells spanning from leukemia to solid tumors [20]. Based on the results of our previous study which showed that the inhibition of PI3Kδ using Idelalisib could induce cytotoxic effects in APL-derived NB4 cells [21], it was tempting to examine whether Idelalisib could enhance the chemo-sensitivity of the cells to ATO. Notably, our data showed that while ATO could reduce viability and hamper NB4 cell metabolic activity, its combination with the increasing concentrations of Idelalisib for 24 and 36 h displayed superior cytotoxicity, indicating that PI3Kδ inhibitor dramatically raised ATO-mediated cytotoxicity in NB4 cells in a time- and concentration-dependent manner. Our results showed that when Idelalisib at the concentrations of 10 and 20 µM are combined with ATO at the concentration of 1 µM, this inhibitor could robustly intensify the anti-leukemic effect of ATO (Fig. 1 A). To elucidate whether the interaction of these agents executed synergistic effects on the growth of NB4 cells, we calculated the combination index (CI). Our results illustrated that the combination of ATO (1 µM) and Idelalisib in all concentrations reduced CI values, indicative of the presence of synergistic effects between the two agents (Fig. 1B).

Fig. 1.

Fig. 1

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The synergistic effects of Idelalisib with ATO in APL-derived NB4 cells. (A) Using trypan blue and MTT assays, the inhibitory effects of Idelalisib/ATO on NB4 cells were determined. Values are given as mean ± S.D. of three independent experiments. * P ≤ 0.05 represented significant changes from the control. (B) The calculation of the combination index (CI) indicated the presence of a synergistic effect between both agents

Inhibitory influence of Idelalisib on ATO-induced anti-proliferative effect on NB4 cells

To investigate whether the combination of ATO (1 µM) with Idelalisib could reduce the number of NB4 viable cells and hinder the ability of the cells to synthesize DNA, both the cell counting and BrdU cell proliferation assay were conducted, respectively. As presented in Fig. 2, our results were suggestive of the enhancive effect of Idelalisib on ATO-induced anti-proliferative effects, as revealed by the more evident reduction in both the number of viable cells (cell growth) and DNA synthesis rate when NB4 cells were treated with both agents as compared with single agents. This finding was also in agreement with the results of cell cycle analysis, which disclosed that in the presence of both agents, the percentage of cells in the S phase was decreased more remarkably, as compared to each agent alone (Fig. 2). In a previous study conducted by Ghaffari et al., the telomerase activity has been suggested to serve as a prognostic factor for a sunset of APL patients with a more aggressive phenotype and those who may not respond well to ATO [22]. Since the telomerase activity is representative of the proliferative capacity of the cells [23] and based on our favorable results indicating the ability of Idelaisib in enhancing the antiproliferative effect of ATO, it was of particular interest to evaluate the combined effect of these agents on the expression of hTERT and c-Myc, as the main regulators of telomerase activity in NB4 cells. The results of real-time PCR analysis revealed that Idelalisib down-regulated the expression levels of both hTETR and c-Myc in ATO-treated cells (Fig. 2); suggestive of the probable promising effects of PI3K inhibition on the therapeutic value of ATO.

Fig. 2.

Fig. 2

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The enhancive effect of Idelalisib on ATO-induced anti-proliferative effect. Simultaneous treatment of Nb4 cells with Idelalisib and ATO resulted in superior anti-proliferative effects, as revealed by the significant reduction in the number of viable cells as well as the ability of the cells to synthesize DNA. Idelalisib-plus-ATO increased the proportion of the cells in the sub-G1 phase of the cell cycle and diminish the percentage of the cells in the S phase. The results of the qRT-PCR analysis showed that the combined treatment strategy had a more vigorous suppressive effect on the expression of hTERT and c-Myc as compared to either drug as a single agent. Values are given as mean ± S.D. of three independent experiments. * P ≤ 0.05 represented significant changes from the control

Idelalisib strengthened ATO-induced cytotoxicity in NB4 through induction of apoptosis

The results of the DNA content analysis showed that when NB4 cells were simultaneously treated with Idelalisib and ATO, the transition of the cells from the sub-G1 phase of the cell cycle was hampered (Fig. 2). To determine whether the stimulatory effects of Idelalisib on ATO-induced cytotoxicity was associated with the induction of apoptosis, the percentage of the cells undergoing apoptosis was evaluated using flow cytometry. Our results demonstrated that co-treatment of the cells resulted in a superior increase in the proportions of both Annexin-V and Annexin-V/PI positive cells (Fig. 3). In order to investigate the molecular mechanisms underlying drugs-induced apoptosis in NB4 cells, the mRNA levels of apoptosis-associated key regulator genes were also investigated. Our results showed that concomitant treatment of the cells with Idelalisib and ATO led to greater changes in the transcription of both pro-and anti-apoptotic genes in comparison with the single agent of ATO. As presented in Fig. 3, we found that ATO-plus-Idelalisib not only elevated the mRNA expression of Bax, but also down-regulated the expression of the anti-apoptotic genes including Bcl-2, MCL-1, survivin, and c-IAP. Our results also indicated that the simultaneous treatment of PI3Kδ inhibitor Idelalisib with ATO resulted in a significant decline in the expression of the p73 gene (Fig. 3).

Fig. 3.

Fig. 3

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Suppression of PI3K using Idelalisib could enhance the apoptotic effect of ATO in NB4 cells. After incubation of NB4 cells with Idelalisib in combination with ATO, the percentages of Annexin-V-positive and Annexin-V/PI-positive cells were increased remarkably as compared to the ATO-treated group. Examination of the expression level of apoptotic target genes revealed that simultaneous treatment with Idelalisib and ATO resulted in more evident alteration in the mRNA level of the genes. Values are given as mean ± S.D. of three independent experiments. * P ≤ 0.05 represented significant changes from the control

Accumulation of ROS is coupled with caspase3-dependent apoptosis

Previous studies showed that there is a reciprocal association between the production of reactive oxygen species (ROS) and the cell sensitivity to ATO, as not only ATO induced its anti-leukemic effects through a ROS-dependent manner [24] but also the production of ROS in malignant cells could increase the responsiveness of the cell to ATO [25]. Noteworthy, we found that PI3K inhibition using Idelalisib promoted ROS production in both myeloid and lymphoid leukemic cells [21]. Of note, our results declared that when the PI3K signaling axis was blocked by Idelalisib, the ability of ROS to increase the production of intracellular ROS was intensified (Fig. 4), indicating that probably Idelalisib promotes the sensitivity of NB4 cells to the apoptotic effects of ATO through ROS-dependent manner. This finding was further confirmed by the results of the caspase-3 assay, as it showed that while single agents of Idelalisib and ATO only minimally augmented caspase-3, simultaneous treatment of NB4 cells with both agents elevated the enzymatic activity of the enzyme up to 6.4 fold (Fig. 4).

Fig. 4.

Fig. 4

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The effect of Idelalisib-plus-ATO on the production of intracellular ROS and caspase-3 enzymatic activity. Upon exposure of Nb4 cells to Idelalisib and ATO, either as a single agent or in a combined modality, there was a significant elevation in the intracellular levels of ROS as well as the enzymatic activity of caspase-3. Values are given as mean ± S.D. of three independent experiments

Suppression of autophagy using CQ could potentiate the anti-leukemic effects of Idelalisib-plus-ATO on NB4 cells

Class I PI3K not only regulates many physiological functions such as cell growth but also may influence autophagy, which serves a dual role in either cell survival or death [26]. To detect the effect of autophagy in the induction of cell death in NB4, APL-derived NB4 cells were treated with various concentrations of autophagy inhibitor chloroquine. Interestingly, we found that the inhibition of autophagy using CQ, as revealed by a conspicuous reduction in the red-to-green fluorescence intensity ratio, decreased cell viability of APL-derived cells in a concentration-dependent manner (Fig. 5). To explore whether inhibition of the autophagy system in NB4 cells could change the responsiveness of the cells to the anti-leukemic effect of Idelalisib, ATO, and the combination of both drugs, we treated the cells with the cocktail consisting of CQ, Idelalisib, and ATO. Intriguingly, we found that treatment of the cells with 25 µM of CQ in combination with ATO and/or Idelalisib exerted superior cytotoxicity as compared to each agent alone (Fig. 5). Taken together, the resulting data suggested that the activation of autophagy in response to either Idelalisib or ATO may at least partly contribute to the acquisition of chemo-resistant phenotype in APL cells.

Fig. 5.

Fig. 5

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Suppression of autophagy using CQ could potentiate the anti-leukemic effects of Idelalisib-plus-ATO on NB4 cells. NB4 cells were treated with an autophagy inhibitor (CQ) for 24 h and visualized by acridine orange staining under a

fluorescence microscope. After incubating NB4 cells with the increasing concentrations of CQ for 24 h, the cytotoxic effect was assessed using the trypan blue exclusion assay. Simultaneous treatment of NB4 cells with a non-cytotoxic concentration of CQ (25 µM) in combination with Idelalisib and/or ATO resulted in superior cytotoxicity. Values are given as mean ± S.D. of three independent experiments. * P ≤ 0.05 represented significant changes from the control

Discussion

The incidence of drug resistance is a common event in patients suffering from leukemia, in particular acute promyelocytic leukemia (APL), and the number of drugs that are incorporated into the treatment regimen of this malignancy are increasing, confronting patients with serious complications of drugs side effects. For a while, the identification of the therapeutic value of arsenic trioxide (ATO) in the treatment of APL has revolutionized the prognosis of the disease and brought promising results for patients who were refractory to ATRA and other chemotherapeutic agents [27]. However, it did not take long since the dose-related toxicity along with the incidence of ATO resistance restricted the clinical application of this agent [28]. Endorsed attempts have been accomplished thus far to improve the outcome of APL patients; however, much of these efforts were not successful enough due to the heterogeneity of the disease, leading to the aberrant activation of cell signaling pathways [29]. When it comes to hematologic malignancies and the incidence of drug resistance, the PI3K singling axis captures all the attention, as this cascade could orchestrate a wide range of cell responses in the neoplastic cells leading to the maintenance of their survival. In a study conducted by Neri et al., it has been indicated that the activation of the PI3K axis in the HL-60AR cell line resulted in the acquisition of resistance phenotype to a wide range of chemotherapeutic drugs [7]. In another study, the portrait of the PI3K axis in the induction of chemo-resistance, in particular to ATO, became even sharper, as the results declared that ATO was unable to reduce the viability of those cells with elevated phosphorylated Akt [6]. Additionally, it has been suggested that suppression of the PI3K signaling axis using Idelalisib sensitized chronic myeloid leukemia (CML)-derived cell line to the anti-leukemic effects of imatinib; suggestive of the probable role of the axis in orchestrating resistance mechanism against this well-known and potent tyrosine kinase inhibitors that is used as a first-line treatment in CML [30]. In an effort to evaluate whether abrogation of the PI3K axis could reinforce the sensitivity of leukemic cells to the cytotoxic effects of ATO, we designed experiments to examine the synergistic effects between an inhibitor of PI3Kδ and ATO in APL-derived NB4 leukemic cells.

We found that blockage of the PI3K axis in NB4 cells using Idelalisib was prospered to enhance both the cytotoxic and the antiproliferative effects of ATO at the concentration of 1 µM. This finding was in accordance with the results of a previous investigation which showed that abrogation of the PI3K axis not only diminished the survival of CLL-derived cells but also potentiated the therapeutic potential of ATO [31]. The present study also delineated that Idelalisib augments ATO-induced anti-proliferative effects, as evidenced by the decrease in the cell population of the S phase of the cell cycle, the ability of the cells to synthesize DNA, and decreased number of inhibitor-treated viable cells. Consistently, it has also been declared that PI3K inhibition in chronic myeloid cells could also produce synergistic effects for imatinib through induction of G1 cell cycle arrest [30]. In the present study, we found that upon the simultaneous exposure of NB4 cells to Idelalisib and ATO, the percentage of the cells was increased in the sub-G1 phase of the cell cycle, a stage at which cells become highly sensitive to the induction of apoptotic cell death under the influence of external signals. Through down-regulation of the expression of p73 and its associated transcription factor c-Myc [32], many anti-cancer agents could increase the production of intracellular ROS in the neoplastic cells and thereby stimulate the apoptotic pathway [33]. Consistent with this, our data showed that while the single agents of Idelalisib and ATO could minimally elevate the generation of ROS in NB4 cells, the combination of the two agents not only increased the ability of the cells to suppress the expression of both c-Myc and p73 but also increased the production of ROS to the greater extent. Moreover, the combination of both agents provided signaling through which APL-derived NB4 cells underwent caspase-3-dependent apoptosis, which was coupled with the downregulation of anti-apoptotic target genes as well as the up-regulation of Bax (Fig. 6). In accordance with our findings, there are several reports in human leukemia cells suggesting that the stimulation of ROS generation within the neoplastic cells could be recruited as a therapeutic tool to reduce the survival of the cells [34, 35] .

Fig. 6.

Fig. 6

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Schematic representation proposed for the plausible mechanisms by which Idelalisib enhanced the anti-leukemic activity of ATO in NB4 cells. Although suppression of PI3K in NB4 cells or exposure of the cells with ATO, as a single agent could diminish the survival and proliferative capacity of leukemic cells when these agents were used simultaneously their ability to enhance the apoptotic cell death was reinforced more significantly. The resulting data also revealed that the cytotoxic effect of Idelalisib/ATO in NB4 cells was probably mediated through suppression of p73 and c-Myc, which was coupled with the elevation in the intracellular level of reactive oxygen species and induction of caspase-3-dependent apoptotic cell death

One of the main advantages of leukemic cells to restrict the success of treatment and thwart all efforts to achieve a complete remission is their ability to at once trigger a compensatory mechanism that can reduce the effectiveness of anticancer drugs [36]. Autophagy has long been accused of its compensatory action in acute myeloid leukemia; however, the paradoxical portrait of this system in the maintenance of leukemic survival or eradicating the neoplastic cells resulted that this system does not being fully convicted and still is under evaluation [37]. Of note, the results of the present study declared that the treatment of NB4 cells with autophagy inhibitor CQ decreased the viability of cells either as a single agent or in combination with ATO and/or Idelalisib; clarifying the possible role of autophagy in the survival maintenance of leukemic cells. Similar findings were reported in the previous investigations, which shed light on the mitigating effects of autophagy on the cytotoxic effects of novel small molecule inhibitors of PI3K [19]. Hitherto, roles deviating from the canonical activity of hTERT have been demonstrated and it became evident that this main regulator of telomerase activity could also facilitate the stimulation of autophagy in cancer cells [38]. Interestingly, the suppression of the PI3K axis in NB4 cells significantly enhanced the inhibitory effect of ATO on the expression of hTERT, reflecting more light on the valuable adjuvant effects that Idelalisib could add to the treatment protocol of APL (Fig. 6). On aggregate and based on the safety profile of idelalisib, the results of the present study proposed PI3K inhibition as an effective approach to enhance the anti-leukemic effect of ATO in NB4 cells, while reducing its toxic concentrations. However, based on the probable side effects of both agents, further studies are required to evaluate the tolerance of the combination of Idelalisib and ATO in APL patients.

Acknowledgements

The authors would like to express their gratitude to Shahid Beheshti University of Medical Sciences (Tehran, Iran) for supporting this study.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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