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Year : 2017  |  Volume : 13  |  Issue : 3  |  Page : 544-549

Honey bee venom combined with 1,25-dihydroxyvitamin D3as a highly efficient inducer of differentiation in human acute myeloid leukemia cells

1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
2 Department of Biology, Faculty of Sciences, Malayer University, Malayer, Iran

Date of Web Publication31-Aug-2017

Correspondence Address:
Maryam Rahimi
Department of Biology, Faculty of Sciences, Malayer University, P. O. Box: 65719-95863, Malayer
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.183220

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 > Abstract 

Purpose: Most cancer cells exhibit a defect in their capacity to mature into nonreplicating adult cells and existing in a highly proliferating state. Differentiation therapy by agents such as 1,25-dihydroxyvitamin D3(1,25-(OH)2 VD3) represents a useful approach for the treatment of cancer including acute myeloid leukemia. Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-(OH)2 VD3. However, usage of these findings in the clinical trials is limited by calcemic effects of 1,25-(OH)2 VD3. Attempts to overcome this problem have focused on a combination of low concentrations 1,25-(OH)2 VD3 with other compounds to induce differentiation of HL-60 cells. In this study, the effect of honey bee venom (BV) and 1,25-(OH)2 VD3, individually and in combination, on proliferation and differentiation of human myeloid leukemia HL-60 cells were assayed.
Materials and Methods: In this in vitro study, toxic and nontoxic concentrations of BV and 1,25-(OH)2 VD3 were tested using Trypan blue stained cell counting and (3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In addition, differentiation of cells was assayed using a Wright-Giemsa staining and nitroblue tetrazolium reduction test. Data were analyzed by a one-way analysis of the variance test using SPSS software.
Results: Our findings showed that both the BV and 1,25-(OH)2 VD3, in a dose and time-dependent manner, caused cell death at high concentrations and inhibited cell proliferation at lower concentrations. About 5 nM of 1,25-(OH)2 VD3 induced differentiation of HL-60 cells to monocytes after 72 h. 2.5 μg/ml of BV suppressed proliferation of HL-60 cells but had not any effects on their differentiation, whereas in combination with 5 nM of 1,25-(OH)2 VD3, it enhanced antiproliferative and differentiation potency of 1,25-(OH)2 VD3.
Conclusions: These results indicate that BV potentiates the 1,25-(OH)2 VD3-induced HL-60 cell differentiation into monocytes

Keywords: 1,25-dihydroxyvitamin D3, differentiation therapy, honey bee venom, human myeloid leukemia HL-60 cells

How to cite this article:
Mohseni-Kouchesfahani H, Nabioni M, Khosravi Z, Rahimi M. Honey bee venom combined with 1,25-dihydroxyvitamin D3as a highly efficient inducer of differentiation in human acute myeloid leukemia cells. J Can Res Ther 2017;13:544-9

How to cite this URL:
Mohseni-Kouchesfahani H, Nabioni M, Khosravi Z, Rahimi M. Honey bee venom combined with 1,25-dihydroxyvitamin D3as a highly efficient inducer of differentiation in human acute myeloid leukemia cells. J Can Res Ther [serial online] 2017 [cited 2022 Jul 1];13:544-9. Available from: https://www.cancerjournal.net/text.asp?2017/13/3/544/183220

 > introduction Top

Most cancer cells display a defect in their capacity to differentiate from nonreplicating adult cells, thus existing in a highly proliferating state. Current attempts to reduce the number of these immature cells include chemotherapy, radiotherapy, and surgery. Chemotherapy is also cytodestructive for normal cells, and some leukemia cells develop resistance to chemotherapy and are therefore difficult to destroy. Differentiation therapy, with the goal of arresting the growth of malignant cells by inducing normalization of cell phenotype without damage to normal tissues, provides an alternative strategy for the treatment of several types of neoplastic diseases. The induction of terminal differentiation by usual antineoplastic agents represents an interesting approach to the treatment of cancer. Since cells exposed to these inducer agents do not endure the cytodestruction produced by cytotoxic agents, instead they acquire the phenotypic characteristics of final-stage adult cell forms with no replicative capacity and ultimately undergo programmed cell death.[1],[2],[3],[4] Acute myeloid leukemia (AML), commonly called AML, is a type of malignant bone marrow, in which there is a lack of mature blood cells in the myeloid line of cells and an excess of immature cells (myelocytes). The HL-60 cell lines are originated from a patient with AML and have been frequently employed as an excellent model line for the in vitro studying of cellular differentiation because of their property that allows the differentiation into either monocytic or granulocytic lineages according to inducing stimuli.[5],[6] Studies have shown that high level of protein kinase C (PKC) has a positive effect on HL-60 differentiation along the monocyte pathway and also cell differentiation was notably inhibited by PKC inhibitors.[7] 1,25-(OH)2 VD3 is known to act as a powerful differentiation inducer in various types of cancer cells including AML cells. However, supraphysiological concentrations of 1,25(OH)2 VD3 required to induce terminal maturation of AML cells can cause lethal hypercalcemia in vivo.[7],[8],[9] Beside, challenges to these aims inducing differentiation in HL-60 cell lines with 1,25(OH)2 VD3 can be enhanced by means of some agents which inhibit nuclear factor-kappa B (NF-κB).[10] A series of papers shown that some materials of antioxidants and anti-inflammatory can enhance the ability of 1,25(OH)2 VD3 in cell differentiation. For example, carnosic acid, silibinin, Tanacetum parthenium extracts, and curcumin effectively potentiate 1,25(OH)2 VD3 and induced cell differentiation in vitro[11],[12],[13],[14] and extended the life span of animal models inoculated with murine leukemia.[15],[16] Similar effects of enhanced differentiation could be obtained using everolimus, immunosuppressant used in transplantation medicine and in oncology, which inhibits mammalian target of rapamycin.[17] Interestingly, inhibitors of p38 kinases α and β,[18] as well as inhibitors of phospholipase A2[19] and nonspecific inhibitors of cyclooxygenase (COX)[20] do the same. Bee venom (BV) therapy may give new hope for treating some diseases such as arthritis, rheumatism, back pain, skin diseases, and cancer.[21],[22] BV can cause growth arrest and cytotoxic effects on numerous types of cancers.[23],[24],[25],[26],[27],[28] Apitoxin or BV is a colorless liquid consisting of a complex mixture of proteins such as melittin, apamin, and adolapine, enzymes including hyaluronidase and phospholipase A2, and biologically active amines such as histamine and epinephrine as well as nonpeptide components with numerous medicinal properties.[21],[22] Researches from the early 1980s until the present have shown that BV has anticancer and anti-inflammatory activities.[22],[29],[30],[31],[32] In this study, we investigate the synergistic effect of BV and 1,25(OH)2 VD3 on proliferation and differentiation of HL-60 cell line.

 > Materials and Methods Top

Bee venom collection

Iranian honey bee (Apis mellifera) venom was prepared by placing bees on a 6-mm wire grid, which was electrically pulsed. The bees then produced venom that dropped on to a glass slide, from which it was collected and freeze-dried according to the method of Lariviere and Melzack.[33]

Preparation of differentiation inducers

One millimeter stock solution of 1,25-(OH)2 VD3 was dissolved in absolute ethanol (Hayman Limited, U.K.) and stored at −80°C. BV was dissolved in dimethylsulphoxide (DMSO), for making a 1 mg/ml stock solution. The solutions were diluted at in the growth medium such that the final concentration of ethanol and DMSO had no effect on the differentiation and proliferation of HL-60 cells. All the sample manipulations were carried out in subdued light.

Cell culture

The human myelocytic leukemia cells (HL-60 cells) were purchased from the Pasteur Institute (Tehran, Iran). Cells were maintained in RPMI-1640 medium (Gibco, UK) and supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, 100 μg/mL (Gibco) streptomycin, in a humidified incubator filled with 5% CO2 at 37°C. The medium was replaced every 48 h.

Assessing cytotoxic activity

To determine the cytotoxic effects of BV separately and in combination with 1,25-(OH)2 VD3 complex on the HL-60 cells, cell viability was tested using (3 [4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) assay. The cells were first seeded in 96-well-cell culture plates (Nunc, Denmark) at a density of 3 × 104 cells/mL/well and subsequently incubated in a humidified 5% CO2 environment, for 1 h. The cells were then treated with 0.5, 1, 2, 2.5, 5, 7.5, and 10 μg/mL BV, and separately with 1,25-(OH)2 VD3 at 1, 5, 25, and 50 nM for 24.,48, and 72 h. The concentrations were chosen as a result of precipitation of the BV in the medium. Nontreated cells were used as controls. In brief, after each treatment, 100 μL of 5 mg/mL MTT (Sigma, Aldrich, MO, USA) was added to each well in 96-well plates. After incubation for 4 h at 37°C, the crystals of viable cells were solubilized by acid-isopropanol (100 ml of 0.04 N HCl in isopropanol). After an overnight incubation in darkness, the optical density (O.D.) of each well was measured at a wavelength of 570 nM using a spectrophotometer. The O.D. values of the experimental groups were divided by those of the untreated control group, and the results were presented as the percentage of cell viability.

Morphological analysis

To monitor the effect of BV alone and in combination with 1,25-(OH)2 VD3 complex on HL-60 cells, the cells were treated with BV and BV/1,25-(OH)2 VD3 complex, then morphologically analyzed under an inverted microscope. Morphological studies of single-cell suspensions were prepared and 2 × 105 cells were loaded into a cytofunnel and centrifuged at 27 g in a cytospin centrifuge. The slides were fixed with methanol and dried. The slides were stained with Wright-Giemsa (sigma) stain solution for 20 min and rinsed in deionized water, air dried, and observed under a microscope with a camera. The stained cells were assessed for size, regularity of the cell margin, and morphological characteristics of the nuclei.

Nitroblue tetrazolium reduction assay

HL-60 cell differentiation was assessed by the nitro blue tetrazolium (NBT) reduction assay, as previously described.[5] The assay is based on the ability of phagocytes to produce superoxide upon stimulation with tissue plasminogen activator (TPA). For this assay, 2 × 105 cells were harvested via centrifugation and then incubated with an equal volume of 0.2% NBT (Sigma) dissolved in phosphate-buffered saline containing 1 ng/ml of freshly diluted TPA at 37°C for 30 min in the dark. Cytospin slides were prepared and examined for blue-black nitroblue formazan deposits, an indicative of a TPA-stimulated respiratory burst. At least 300 cells were assessed for each experiment.

Statistical analysis

Student's t-test and one-way analysis of the variance were used to determine the statistical significance of differences between values for various experimental and control groups. P < 0.05 was considered statistically significant.

 > Results Top

Proliferation assay of HL-60 cell line

Cell proliferation assay with trypan blue exclusion assay and the MTT assay showed that HL-60 cell lines are highly proliferative. In this setting, mean doubling time of HL-60 cell line was 22.5 h.

Effect of bee venom and 1,25-dihydroxyvitamin D3 on the cell proliferation of HL-60

As shown in [Figure 1]a, BV in concentrations over 10 μg/ml destroyed all of the cells immediately and in concentrations of 2.5–10 μg/ml in a dose and time-dependent manner caused cell death and in concentrations of 2.5 μg/ml inhibited cell proliferation but in concentrations under 2.5 μg/ml did not have any effects on the cells. Furthermore, results showed that levels between 25 and 50 nM of 1,25-(OH)2 VD3 were very toxic, but concentrations between 1 and 5 nM were nontoxic and did not cause cell death although caused inhibition of cell proliferation of HL-60 cell line [Figure 1]b. In addition, the evaluation of the synchronic effect of inhibitory concentration of BV and 1,25-(OH)2 VD3 on the proliferation of HL-60 cell lines, demonstrated a significant upregulation in inhibition of cell proliferation compared to the use of inhibitory concentrations of them alone in 72 h [Figure 2].
Figure 1: Effect of bee venom and 1,25-dihydroxyvitamin D3 on the viability of HL-60 cells: HL-60 leukemia cells were treated with various concentrations of bee venom for 24, 48 and 72 h (a) and various concentrations of 1,25-dihydroxyvitamin D3 for 24-48-72 h (b) and percentage of viable cells was determined by (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The experiment was repeated more than 3 times with similar results indicate significant difference (P < 0.05) between specified time and the previous time period

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Figure 2: Effect of bee venom and 1,25-dihydroxyvitamin D3 in combination with together on HL-60 cell proliferation: HL-60 leukemia cells were treated with combination of 2.5 μg/ml of bee venom, and 5 nM of 1,25-dihydroxyvitamin D3 and percentage of viable cells/control was determined by (3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The experiment was repeated more than 3 times with similar results. * and denote statistically significant difference (P < 0.001) compared to control cells, cells that treated with 2.5 μg/ml of bee venom and cells that treated with 5 nM of 1,25-dihydroxyvitamin D3, respectively

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Functional and morphological studies of HL-60 cells treatment with honey bee venom and 1,25-dihydroxyvitamin D3

To determine the effect of BV on 1,25-(OH)2 VD3-induced cell differentiation, the HL-60 cells were treated with inhibitory concentration of BV (2.5 μg/ml) and 1,25-(OH)2 VD3(5 nM). Then, the number of differentiated cells were determined by NBT reduction assay and Wright-Giemsa stain. As controls, the cells were treated with either 1,25-(OH)2 VD3 or BV alone. NBT assay and Wright-Giemsa stain showed that the addition of BV to cultures exposed to a suboptimal concentration of 1,25-(OH)2 VD3(5 nM), which by itself caused a relatively low level of differentiation (<22%), resulted in a marked increase in the degree of cell differentiation. BV by itself did not induce significant cell differentiation (<5% of the cells attained a differentiated phenotype) [Figure 3]. As shown in [Figure 4], Wright-Giemsa stained undifferentiated HL-60 control cells were predominantly myelocytes with round and regular cell margins, and large nuclei, suggesting that the cells were highly active in DNA synthesis and were rapidly proliferating. Combined treatment of HL-60 cells with 1,25-(OH)2 VD3 and BV resulted in significantly decreased cell size and increased chromatin density and also increased cytoplasmic-nuclear ratio, which suggested less DNA synthesis. Some cells showed a horseshoe-shaped nucleus, which is a sign of cell differentiation into a monocytic lineage.
Figure 3: Effects of bee venom alone and in combination with 1,25-dihydroxyvitamin D3 on HL-60 leukemia cell differentiation: HL-60 cells were treated for 72 h with 2.5 μg/ml of bee venom and 5 nM of 1,25-dihydroxyvitamin D3 alone or in combination for 72 h and percentage of differentiated cells to monocyte/control was evaluated via Wright-Giemsa staining and nitro blue tetrazolium reduction assay. indicate significant difference (P < 0.001) of differentiated cells to monocyte/control

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Figure 4: Morphologic analysis of HL-60 cells treated with 2.5 μg/ml of bee venom in combination with 5 nM of 1,25-dihydroxyvitamin D3: HL-60 cells were treated for 72 h with 2.5 μg/ml of bee venom plus 5 nM 1,25-dihydroxyvitamin D3. Cytospin slides were made from HL-60 cells (5 × 105 cells ml−1) and were stained with wright-Giemsa stain (I) undifferentiated cells, (II, III, IV) differentiated cells to monocytes. Scale bar: 100 μm

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 > Discussion Top

In this study, we have demonstrated that BV and 1,25-(OH)2 VD3 in time- and dose-dependent manner in high concentration caused cell death and in low concentration inhibited cell proliferation. BV in 5 μg/ml and 7.5 μg/ml concentrations during 48 h killed 50% (85%, respectively) of cells. 1,25-(OH)2 VD3 in the concentration of 25 nM in 72 h destroyed 50% 0f cells while upper concentrations killed higher percentage of the cells. In addition, we observed that 2.5 μg/ml of BV and 5 nM of 1,25-(OH)2 VD3 in 72 h without cytotoxic effects inhibited cell proliferation. In line with our findings, previous studies have reported 5 nM 1,25-(OH)2 VD3 as nontoxic and inhibitory concentration,[8],[34],[35],[36] Moreover, we for the 1st time showed that BV potentiated 1,25-(OH)2 VD3-induced differentiation of HL-60 myelocytic leukemia cells into a monocytic lineage although BV alone did not induce the cell differentiation. Previous studies have shown that 1,25-(OH)2 VD3 may mediate biological responses including cell differentiation as a consequence of a genomic pathway signaling through the Vitamin D receptor to modulate gene transcription[37] and of a nongenomic pathway through a putative cell membrane receptor to generate rapid effects[38] including the opening of voltage-gated calcium and chloride channels,[39] and activation of PKC and MAPK.[40],[41] Furthermore, previous reports showed 1,25-(OH)2 VD3 as a cell differentiation agent via inhibition of NF-κB activity (a genomic pathway of 1,25-(OH)2D3-induced differentiation). However, 1,25-(OH)2 VD3 cannot be used continuously, due to the occurrence of hypercalcemia. Inducing differentiation in HL-60 cell lines with 1,25-(OH)2 VD3 can be enhanced by the means of some interferencing agents. Previous studies have reported some chemicals were capable of enhancing the differentiation of HL-60 cells produced by low levels of 1,25-(OH)2 VD3. These include butyrate,[42] capsaicin,[43] silibinin,[44] magnolol and honokiol,[45] indirubin,[34] artemisinin,[46] parthenolide, and nonsteroidal anti-inflammatory agents such as acetylsalicylic acid.[37] Based on studies performed in this research, we showed that BV potentiated 1,25-(OH)2 VD3-induced differentiation of HL-60 cell lines. However, the mechanism by which BV potentiates 1,25-(OH)2 VD3-induced HL-60 cell differentiation is not clear. Based on our knowledge, agents that were capable of enhancing the differentiation potential of 1,25-(OH)2 VD3, activated one or more signaling pathways. For example, acetylsalicylic acid as nonsteroidal anti-inflammatory agents and parthenolide via inhibition of NF-κB activity, artemisinin by activation of PKC, nonsteroidal anti-inflammatory agents via inhibition of COX and increasing CAMP in cells were capable of enhancing the differentiation potential of 1,25-(OH)2 VD3 to monocyte linage.[20],[35],[37],[46] Previous studies showed that BV with active compounds such as melittin and phospholipase A2 were capable of inhibiting the COX and NF-κB activity.[22],[29],[30],[31],[32]

 > Conclusions Top

It seems that BV was capable of enhancing the differentiation potential of 1,25-(OH)2 VD3 via inhibiting of COX and NF-κB activity. Further studies using in vivo models are needed for applicability of BV and 1,25-(OH)2 VD3 in the clinical settings.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

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