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Kermanian propolis induces apoptosis through upregulation of Bax/Bcl-2 ratio in acute myeloblastic leukemia cell line (NB4)

1 Department of Hematology and Laboratory Sciences, Faculty of Allied Medical Sciences; Cell Therapy and Regenerative Medicine Comprehensive Center, Kerman University of Medical Sciences, Kerman, Iran
2 Department of Traditional Medicine, Faculty of Traditional Medicine, Kerman University of Medical Sciences, Kerman, Iran
3 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Cell Therapy and Regenerative Medicine Comprehensive Center; Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, Iran

Date of Submission07-Jul-2021
Date of Decision03-Jan-2022
Date of Acceptance22-Jan-2022
Date of Web Publication23-Nov-2022

Correspondence Address:
Alireza Farsinejad,
Cell Therapy and Regenerative Medicine Comprehensive Center, Kerman University of Medical Sciences, Kerman
Reza Vahidi,
Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_1084_21

 > Abstract 

Objective: Propolis is a viscous resinous honeybee-produced substance with numerous medicinal functions; its composition and texture varies according to the geographic location. It is considered to be a promising natural source for the management and prevention of various pathological conditions. Although several studies have exhibited the anti-cancer activity of different types of propolis, the tumor-suppressing potential of Kermanian propolis against leukemia cell lines has remained poorly understood. Therefore, the current experiment was aimed to reveal the anti-tumor activity of this bioactive compound both as monotherapy and combined therapy with cytarabine against an acute myeloid leukemia (AML) cell line, NB4.
Materials and Methods: Following the treatment of NB4 cells with either Kermanian propolis (5, 10, 20, 40, 80, 160, and 320 μg/mL), cytarabine (0.1, 0.25, 0.5, 0.75, 1, and 2 mM), or their combination (40 and 80 μg/mL of Kermanian propolis along with 0.1, 0.25, and 0.5 mM of cytarabine), colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to measure the viability (%) of the cells. Next, to examine the apoptotic rate and the pattern of corresponding gene expression (Bcl-2, Bax, p53, and p21), Annexin-V/PI staining by flow cytometry and quantitative Real-Time polymerase chain reaction assays were performed, respectively.
Results: We perceived significant apoptosis induction in a dose-dependent manner following the treatment with Kermanian propolis, cytarabine, and also their combination in the NB4 cell line. In addition, the combined treatment was associated with lower expression of the anti-apoptotic gene (Bcl-2) and higher expression of the pro-apoptotic genes (p53, Bax, and p21) in comparison to mono treatments.
Conclusion: The synergistic anti-tumor activity induced by the combination of Kermanian propolis and cytarabine presents a novel and encouraging option for AML treatment.

Keywords: Acute myeloid leukemia, Bax/Bcl-2 ratio, cytarabine, kermanian propolis, NB4

How to cite this URL:
Salavatipour MS, Kouhbananinejad SM, Lashkari M, Bardsiri MS, Moghadari M, Kashani B, Farsinejad A, Vahidi R. Kermanian propolis induces apoptosis through upregulation of Bax/Bcl-2 ratio in acute myeloblastic leukemia cell line (NB4). J Can Res Ther [Epub ahead of print] [cited 2022 Dec 9]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=361900

 > Introduction Top

Acute myeloid leukemia (AML), an aggressive form of hematopoietic malignancies, is caused by uncontrolled proliferation and blocked differentiation of clonal myeloid progenitor cells which take refuge within different tissues, especially in the bone marrow and blood.[1] AML is known as the most common acute leukemia subtype in the USA with an annual incidence rate of 4.3 per 100,000.[2] Although AML could occur in any age group, it is more common in elderly individuals, so that nearly 75% of AML patients aged 65 years or older in the USA.[3] Predominantly, three-step chemotherapy using anthracyclines and cytarabine has been chosen as the standard remedy for this neoplasm.[4] The mechanism of action[5] of cytarabine (1-β3-D-arabinofuranosyl cytosine), as the most effective treatment, is interfering with DNA synthesis through the induction of DNA damage at the S phase and the inhibition of essential enzymes for DNA synthesis (DNA/RNA polymerases and nucleotide reductase). Despite approximately 70% initial remedial effectuality, a high rate of relapse and significant dose-dependent toxicity are the largest impediments toward broad clinical application of cytarabine.[4] Therefore, identifying new strategies aimed to overcome drug resistance, prevent relapse, decrease toxicity, and improve the efficacy of the treatment seems to be necessary. In this regard, the application of natural substances as adjunct therapy has attracted a lot of attention.[6] Propolis (bee glue), a natural gummy and balsamic substance, is prepared by honeybees following mixing their saliva with the resin of flowers, leaves of trees, and plants.[7],[8],[9],[10] Owing to its low toxicity, relative safety, and noticeable biological activities such as anticancer, antioxidant, anti-inflammatory, antibiotic, and antifungal properties, propolis has recently been highly regarded.[11] These biological potentials could vary according to the chemical structure and particularly to the phenolic compounds, which are in turn related to the production area, genetic diversity in the queen bee, the used solvents for extraction, and the season of propolis production.[12] Notwithstanding its varied composition, propolis has gained acceptance as an adjunct treatment option for cancer, since its anti-tumor effects were confirmed by a plethora of studies.[13],[14],[15],[16],[17] However, no study has yet examined the effects of Kermanian propolis against hematologic neoplasms. Considering this shortage of knowledge and also the high prevalence of AML, the focus of the current experiment was to disclose the cytotoxic potential of this bioactive compound (as monotherapy and polytherapy with cytarabine) against the AML cell line NB4.

 > Materials and Methods Top

Substances and drugs

To prepare Kermanian propolis (source: Kerman, Iran) extract, following certifying its character by the Department of Pharmacognosy, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran, the substance was washed three times, dried, squashed, and finally incubated (3 days) with methanol (500 mL, 37°C; Merck). After this period, the methanolic extract was collected at fixed intervals and evaporated by a rotary evaporator (Heidolph). A stock solution was prepared by dissolving an appropriate amount of dehydrated extract (0.016 gram) in 10 μL dimethyl sulfoxide (DMSO, Sigma Aldrich). Then, the final volume was set at 10 mL using a complete cell culture medium. Under sterile conditions, the stock solution was filtered using a 0.22-μm filter (Orange Scientific Company) and then suitable working concentrations were prepared using the culture media. To achieve the required concentrations of cytarabine (Sigma-Aldrich), the appropriate volume of the stock solution (2 mg/mL) was added to the cell culture medium.

Cell culture

The AML cell line (NB4; NCBI code: C515) was obtained from the Iranian cell bank of Pasteur Institute, Tehran, Iran was cultured (37°C, 5% CO2) in RPMI 1640 medium (Gibco) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS, Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Sigma Aldrich). The culture media was replaced every 2 days until cells reached 80%–90% confluency.

Cell viability evaluation

To evaluate the effects of the studied compounds on the overall viability of NB4 cells, MTT colorimetric assay was employed.[18],[19] 2 × 105 cells/well were seeded into a 96-well culture plate containing 0.1 mL of complete culture medium, and desired concentrations of either Kermanian propolis (5, 10, 20, 40, 80, 160, and 320 μg/mL), DMSO-solved cytarabine (0.1, 0.25, 0.5, 0.75, 1, and 2 mM), or their combination (0.1, 0.25, and 0.5 mM for cytarabine and 40 and 80 μg/mL for Kermanian propolis) were used as treatment. In addition to the untreated control, DMSO (0.1%)-treated cells were investigated as a negative control. Following the incubation durations (24, 48, and 72 h), the cells were incubated with MTT solution (5 mg/mL; Sigma-Aldrich) for 4 h at 37°C. Afterward, the plate was centrifuged (700 × g/10 min), the MTT solution was eliminated, and DMSO (100 μL) was added to each well. Finally, the optical density of the wells was measured at 570 nm using the ELISA reader ELX808 device (BioTek), cell survival was analyzed according to the MTT assay kit instruction (Sigma Aldrich) and following formula, and the half-maximal inhibitory concentrations (IC50s) were determined. Furthermore, to confirm the specificity of Kermanian propolis for cancer cells, normal human peripheral blood mononuclear cells (PBMCs) were treated with the same IC50 concentrations.

Determination of combination index value

The combination cytotoxic effect of Kermanian propolis and cytarabine was assessed by CompuSyn software version 1.0 (ComboSyn, Inc., Paramus, NJ, USA) using MTT assay results.[20] This software calculates the Combination Index (CI) value by the median effect principle. The employed equation is (CI = [D] 1/[Dx] 1 + [D] 2/[Dx] 2), where (Dx) 1 and (Dx) 2 refer to the concentrations of compounds 1 and 2 in a combination needed to achieve the same efficiency as that of compounds when used alone. The CI value <1, = 1, and >1 demonstrate synergism, additive effect, and antagonism, respectively [Table 1].
Table 1: The combination index value and dose-reduction index for the combination of Kermanian propolis and cytarabine

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Apoptosis assay

To investigate the effect of the aforementioned compounds on programmed cell death, the appearance of anionic phosphatidylserine (PS) on the surface of the cells was assayed by the Apo FlowEx® FITC kit (ExBio, ED7044). In brief, cells (10 × 104 cells/well) were seeded into 12-well cell culture plates and exposed with elected concentrations of the compounds for 48 h. Then, the cells were collected, washed with phosphate-buffered saline, and incubated (15 min) with binding buffer, propidium iodide (PI), and Annexin V according to the manufacturer's instructions.[21],[22] Using the CyFlow® Space flow cytometer (Sysmex Partec), the percentage of apoptotic cells (Annexin V+/PI and Annexin V+/PI+ cells) was detected. It should be noted that unlabeled cells were applied to remove autofluorescence and all trials were repeated three times.

RNA extraction and synthesis of cDNA

To evaluate the effect of Kermanian propolis, cytarabine, and Kermanian propolis + cytarabine on the expression of apoptotic genes, cDNAs of the control and treated NB4 cells were subjected to the real-time polymerase chain reaction (PCR) assay. For this purpose, after 48 h of treatment, total RNA was extracted through TriPure Isolation Reagent (Roche Molecular Biochemical). Following the assessment of the purity and integrity of RNA samples using spectrophotometry (A260/A280 ratio) and agarose gel electrophoresis (1.5%), cDNA was synthesized based on the Primescript RT reagent instruction (PrimeScript 1st strand cDNA Synthesis Kit, RR037A). Then, the analysis of gene expression (Bax, Bcl-2, p53, and p21) was conducted. After mixing real Q Plus 2 × Master Mix Green (Amplicon, 5 μL), 1 μL of the cDNA product, 0.5 μL of each primer (10 pmol), and 3 μL of deionized water, the mixture was heated according to Rotor-Gene 6000 Real-time PCR System (Corbett Research) thermal program: initial activation (95°C for 15 min), denaturation (95°C for 15 s), and combined annealing/elongation (60°C for 60 s). Finally, the gene expression value was calculated using the comparative Ct (2−ΔΔCT) method.[23] All trials were performed three times, and sequences of used primers are listed in [Table 2]. Besides, β-actin was used as the internal control.
Table 2: List of primers for the studied genes

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Statistical analysis

After determining the mean ± standard deviation (mean ± SD), the statistical differences between groups (flow cytometry and real-time PCR) were analyzed by the t-test using the SPSS 20 software. On the other hand, MTT results' analysis was executed using two-way analysis of variance. P ≤ 0.05 were regarded as significant, statistically.

 > Results Top

Kermanian propolis reinforces the cytotoxicity of cytarabine

The metabolic activity (an indicator of cell viability) of the NB4 cells after exposure to several concentrations of Kermanian propolis and cytarabine, alone and in combination, was assessed by MTT colorimetric assay. As presented in [Figure 1]a and [Figure 1]b, the metabolic activity of the cells substantially decreased in a dose- and time-dependent fashion (P < 0.05). Based on the results, the 48 h treatment was chosen as the optimum exposure time for the following experiments; the 50% decreases in cell viability (IC50s) in this condition were 80 μg/mL and 0.5 mM for Kermanian propolis and cytarabine, respectively. Furthermore, no significant difference was seen between untreated and DMSO-treated cells (P > 0.05). Besides, treatment of PBMCs with IC50 concentration of Kermanian propolis induced no dramatic apoptotic effect [P > 0.05, [Figure 1]d]. This finding recommends the tumor-selective apoptotic effect of Kermanian propolis.
Figure 1: The combination strategy possessed higher cytotoxicity in NB4 cells compared to the monotherapy approach. NB4 cells were treated with various concentrations of Kermanian propolis (a) and cytarabine (b) for 24, 48, and 72 h and also Kermanian propolis + cytarabine for 48 h (c). The metabolic activity (%) of treated cells was determined relative to DMSO-treated cells (Cnt) which was set as 100% (mean ± standard deviation, n = 3). Simultaneous application of Kermanian propolis and cytarabine noticeably reduced the IC50 compared to the individual use of either compound. Interestingly, no significant apoptotic effect was observed followinf treatment of peripheral blood mononuclear cells with IC 50 concentration of Kermanian propolis (d). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. P: Kermanian propolis, C: cytarabine

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The combined effects of Kermanian propolis and cytarabine on NB4 cells were assessed by Compusyn software based on the MTT results. According to CI analysis, three combination sets had “synergistic” effects [Figure 2]a which were further confirmed as combination points below the additive line in isobologram analysis [Figure 2]b and [Table 1].
Figure 2: Synergistic effect of Kermanian propolis (40, 80 μg/mL) and cytarabine (0.1, 0.25, and 0.5 mM) using Compusyn software. (a) The combination index versus fraction affect curve of Kermanian propolis and cytarabine combination treatment. Per guidelines, the combination index <1, = 1, and >1 illustrated synergistic, additive, and antagonistic effects, respectively. As shown, some combination doses (Kermanian propolis: 40 μg/mL and cytarabine: 0.1 mM, Kermanian propolis: 40 μg/mL and cytarabine: 0.25 mM, and Kermanian propolis: 40 μg/mL and cytarabine: 0.5 mM) had synergistic effects (combination index < 1). (b) Concentration-normalized isobologram analysis of Kermanian propolis and cytarabine combination. (Dx) 1 and (Dx) 2 refer to the concentration of cytarabine and Kermanian propolis in a combination needed to achieve the same efficiency as that of compounds when used alone. Three combination points were below the additive line and had synergistic effects

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Apoptosis induction in NB4 cells as detected by Annexin V-FITC analysis

To determine the cytotoxic effects of Kermanian propolis, cytarabine, or their combination in NB4 cells, we evaluated the apoptosis index using the Annexin V-FITC/PI staining procedure. Compared to the control group, 48 h incubation of cells with the elected concentrations (Kermanian propolis: 80 μg/mL, cytarabine: 0.5 mM, and Kermanian propolis + cytarabine: 40 μg/mL and 0.1 mM) revealed a significant increase in early and late apoptosis of treated cells (P < 0.01). Noteworthy, the combinational treatment caused a more significant apoptotic effect compared to either monotherapy [Figure 3].
Figure 3: Kermanian propolis significantly induced apoptosis in NB4 cells. The level of Annexin V and PI positivity in 48h-incubated cells with Kermanian propolis (80 μg/mL), cytarabine (0.5 mM), and Kermanian propolis + cytarabine (40 μg/mL + 0.1 mM) was examined by flow cytometer. As represented, the Annexin V+/PI and Annexin V+/PI+ cells increased in the combination therapy compared to either monotherapy. Q1, Q2, Q3, and Q4 indicate PI+, Annexin-V+/PI+, Annexin-V/PI, and Annexin-V+ cells, respectively. One representative experiment among 3 independent ones is illustrated (n = 3, **P ≤ 0.01, and ***P ≤ 0.001)

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The combination of Kermanian propolis and cytarabine was associated with the expression of apoptosis-related genes

To further assess the effects of this approach, the expression changes in Bax, Bcl-2, p53, and p21 genes at the RNA level were compared between the combination-treated NB4 cell line and the control group. According to [Figure 4], after 48 h, a significant increase in Bax (P < 0/001), p53 (P < 0/001), and p21 (P < 0/001) gene expression was observed in combined treatment of Kermanian propolis and cytarabine (40 μg/mL Kermanian propolis + 0.1 mM cytarabine) compared to control, and either Kermanian propolis-or cytarabine-treated groups. In addition, NB4 cells in combination groups showed a significant down-regulation of the Bcl-2 gene (P < 0/001) compared to other groups. In sum, our results confirmed that Kermanian propolis had a potential apoptosis-inducing effect on NB4 cells through increasing the expression of Bax, p53, and p21 genes and down-regulation of the Bcl-2 anti-apoptotic gene.
Figure 4: Bax (a), Bax/Bcl2 rati (c), p21 (d), and p53 (e) genes were over-expressed

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Taken together, the present data propose that Kermanian propolis reduces the viability and growth of NB4 cells through the induction of apoptosis. In some concentrations, the amalgamation of Kermanian propolis and cytarabine augments the anti-NB4 activity compared to the monotherapy approach due to synergistic interactions.

 > Discussion Top

Given that currently available chemotherapies face many hurdles (including high cost and toxicity), the discovery of nontoxic and more effective compounds seems necessary.[8],[24],[25],[26] One of the promising substances with numerous applications in the treatment of various diseases (e.g., gynecological, oral, oncological, and dermatological conditions) is propolis.[8],[13] According to previous studies,[8],[10],[13],[14],[27] therapeutic properties of propolis (i.e., antiseptic, anti-inflammatory, antioxidant, antibacterial, and immunomodulatory effects) are associated with its different components including vitamins, minerals, resin, wax, enzymes, essential oils, pollen, and various organic elements such as esters, flavonoids, terpenes, ß-steroids, aromatic aldehydes, phenolic acids, and alcohols. Despite insufficient knowledge of the precise mechanisms of this compound, it seems that the bioactive feature of propolis (9, 10, and 15) mostly relies on the existence of polyphenols, particularly flavonoids (e.g., quercetin, pinocembrin, acacetin, chrysin, rutin, luteolin, kaempferol, apigenin, myricetin, catechin, naringenin, and galangin). Noticeably, the most important known application of propolis and its derivatives is their inhibitory effects across a wide spectrum of tumors that are due to the induction of apoptosis, mitochondrial outer membrane permeabilization, cell cycle arrest, and modulation of angiogenesis, insulin signaling, oxidative stress, and inflammation.[8],[13]

In this regard, Desamero et al.[17] evaluated the tumor-suppressing effects of the ethanolic ethanolic extract of Philippine stingless bee propolis (EEP) against gastric cancer in vitro/vivo in 2019; they revealed that this compound led to cell cycle arrest at the G0/G1 phase through modulation of cell cycle-related genes. Moreover, another study in 2014 ascertained a time-and dose-dependent apoptotic effect for ethanol extract of Chinese propolis (EECP) on MCF-7 (human breast cancer cell line) and low cytotoxic effect on normal human umbilical vein endothelial cells.[28] Similarly, the cytotoxic action of Turkish propolis was observed during another study in 2016.[16] The results of this study determined a selective anti-tumor effect against A549 (human lung cancer cells) in comparison with normal fibroblast cells that were associated with its ability to induce endoplasmic reticulum stress, apoptosis, and caspase activity. Similarly, another study in 2013[29] delineated the time- and concentration-dependent cytotoxic activity of Indian propolis toward various cancer cell lines (such as breast cancer, colon adenocarcinoma, epithelial colorectal adenocarcinoma, and murine melanoma). A further fascinating result of this experiment was desired antioxidant potential of stingless bee propolis.

Although previous research has shown the anti-neoplastic effects of propolis, the effect of Kermanian propolis against hematological malignancies and the corresponding mechanisms still remain unclear. Consequently, the present experiment was designed to investigate the sensitivity of NB4 cells to this compound. To accomplish this objective, the metabolic activity, cellular apoptosis, and apoptotic genes' expression levels in cells were analyzed, in the treatment and control groups, through the MTT, flow cytometry, and real-time PCR tests, respectively. According to MTT data [Figure 1], Kermanian propolis, cytarabine, and Kermanian propolis + cytarabine progressively reduced cell viability with increasing concentrations, and the IC50 concentrations were determined as 80 μg/mL, 0.5 mM, and 40 μg/mL + 0.1 mM, respectively. It should be noted that Kermanian propolis had no apoptotic effect on PBMCs. Our synergism analysis through Compusyn software indicated that the combination of Kermanian propolis and cytarabine in some doses possessed a synergistic effect [Figure 2]. In consistence, MTT and flow cytometry assays confirmed that Kermanian propolis potentiated the cytotoxicity and apoptosis induction of cytarabine on NB4 cells and reduced the dose of cytarabine required to induce 50% cell death [Figure 1]c and [Figure 4]. Similarly, Salim et al. in 2015[30] used the extract of Egyptian propolis (EEP) in combination with doxorubicin (DOX) and reported enhanced anti-proliferative and apoptosis-inducing effects of the combination therapy in the human prostate cancer cell line (PC3) in comparison to treatment with DOX alone. Rouibah et al.[31] also revealed that the combination of Algerian propolis and DOX synergistically increased cell cycle arrest and apoptosis in the human pancreatic cancer cell line. To compare green and red propolis (G12 and G13),[32] Franchi et al. assessed the in vitro cytotoxic potential of these compounds on different human leukemia cells

(K562, HL60, NB4, Ramos human Burkitt lymphoma, Raji human Burkitt lymphoma, Nalm16, Nalm6, RS4, B15, and REH). Higher cytotoxicity of G13 and different IC50 concentrations (<30 and >20 μg/mL for G12; <20 and >15 μg/mL for G13) were the most important findings of their study about NB4. Unfortunately, these researchers did not appraise the apoptosis-associated mechanisms of these compounds.

In order to identify the probable effects of the aforementioned compounds (as monotherapy and in combination) on the relative expression of the apoptosis-related genes, the expression levels of Bax, Bcl-2, p53, and p21 were estimated. These genes, as members of the apoptotic pathway, are key regulators of cell death. The Bcl-2 family exerts a pivotal role in the fate of the cell through anti-apoptotic and pro-apoptotic effects. A well-known member of this family, the Bcl-2 protein, is located at the outer membrane of the mitochondria and acts as an anti-apoptotic protein to inhibit cellular death. On the other hand, Bax suppresses the activity of Bcl-2 and inhibits cell survival through mitochondrial membrane permeabilization.[33],[34] Furthermore, p21 and p53, two genes involved in the inhibition of cell cycle progression could be appropriate indicators of the cell status.[35] As illustrated in [Figure 3], our findings hinted at the considerable reduction of Bcl-2 expression (b) and remarkable up-regulation of Bax (a), p53 (e), and p21 (d) genes, causing an enhancement of the Bax/Bcl-2 ratio (c) in the treated cells.

In search of possible mechanisms of propolis, a previous study exhibited the telomerase inhibition-dependent apoptosis of T-cell lymphoblastic leukemia (CCFR-CEM) cells after exposure to the ethanolic extract of Turkish propolis.[36] In addition, Cogulu et al. confirmed the decreased expression of human telomerase reverse transcriptase in bone marrow-derived leukemic cells after treatment with 60 ng/mL Turkish propolis.[37] Another fascinating experiment by Eom revealed that dose-dependent inhibition of cell proliferation, apoptosis induction, caspase-3 activation, and increased levels of cytosolic cytochrome c are the main anti-neoplastic mechanisms of Korean propolis against HL-60 cells.[38]

In conclusion, our results unveil that Kermanian propolis could be a promising therapeutic through activating apoptotic signaling pathways in the NB4 cell line. Furthermore, this natural substance can induce a synergistic effect with cytarabine to enhance the outcome of treatment and reduce the concentration. However, further molecular/cellular assessments are required to identify other possible anti-cancer mechanisms of this natural product.

Financial support and sponsorship

This work was supported by the Kerman University of Medical Sciences, Kerman, Iran (grant number: 98000106).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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