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Year : 2018  |  Volume : 14  |  Issue : 3  |  Page : 503-508

Hesperidin inhibits insulin-induced phosphoinositide 3–kinase/Akt activation in human pre-B cell line NALM-6

1 Department of Basic Medical Sciences, National Institute and Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Nutrition, Faculty of Paramedicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3 Department of Biochemistry, Fasa University of Medical Sciences, Fasa, Iran
4 Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
5 Department of Clinical Nutrition and Dietetic, National Institute and Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6 Department of Clinical Nutrition and Dietetic, National Institute and Faculty of Nutrition and Food Technology; Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Web Publication12-Jun-2018

Correspondence Address:
Dr. Sayed Hossein Davoodi
Cancer Research Center, Shahid Beheshti University of Medical Sciences, P.O. Box 1989934148, Tehran, Iran, and Department of Clinical Nutrition and Dietetic, National Institute and Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran
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Source of Support: National Institute of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Conflict of Interest: None

DOI: 10.4103/0973-1482.157323

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 > Abstract 

Context: It has been shown that hesperidin induces apoptosis in NALM-6 cells through inhibition of nuclear factor-kappa B (NF-κB) activation.
Aims: To investigate the effect of hesperidin on inhibition of NF-κB activation through blocking phosphoinositide 3–kinase (PI3K)/Akt pathway as a main target in cancer treatment, in NALM-6 cells.
Materials and Methods: NALM-6 cells were incubated with two concentrations of hesperidin (25, 50 μM) in the presence or absence of insulin (100 nM), as a potent activator of Akt. The cytotoxic activity of hesperidin was determined by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptotic death was measured by ELISA test using cell death detection ELISA Plus kit. To assay the effect of hesperidin on Akt pathway, the phosphorylation levels of Akt, inhibitor of kappa B alpha (IκBα), and glycogen synthase kinase-3 beta (GSK-3β) and expression level of IκB kinase alpha (IKKα) were determined by Western blot analysis.
Results: Hesperidin (both concentrations) significantly reduced cells survival in the presence and absence of insulin compared to untreated cells in a time-dependent manner (P < 0.05). Hesperidin also significantly increased apoptosis in NALM-6 cells even in hyperinsulinemia condition (P < 0.0001). Hesperidin inhibited insulin-induced phosphorylation and activation of Akt, IκBα, and GSK-3β and decreased expression of IKKα.
Conclusion: The results of this study demonstrated that cytotoxic and proapoptotic actions of hesperidin are partly mediated through the suppression of PI3K3/Akt/IKK signaling pathway. So, hesperidin might act as a chemotherapeutic agent by targeting cell survival pathways.

Keywords: Apoptosis, glycogen synthase kinase-3 beta, hesperidin, inhibitor of kappa B, inhibitor of kappa B kinase, nuclear factor-kappa B, phosphoinositide 3–kinase/Akt

How to cite this article:
Shahbazi R, Cheraghpour M, Homayounfar R, Nazari M, Nasrollahzadeh J, Davoodi SH. Hesperidin inhibits insulin-induced phosphoinositide 3–kinase/Akt activation in human pre-B cell line NALM-6. J Can Res Ther 2018;14:503-8

How to cite this URL:
Shahbazi R, Cheraghpour M, Homayounfar R, Nazari M, Nasrollahzadeh J, Davoodi SH. Hesperidin inhibits insulin-induced phosphoinositide 3–kinase/Akt activation in human pre-B cell line NALM-6. J Can Res Ther [serial online] 2018 [cited 2020 Jul 8];14:503-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/3/503/157323

 > Introduction Top

Hesperidin is a glycosylated flavanone which is mainly found in citrus fruits.[1] According to in vivo studies, hesperidin shows different biological effects such as anti-inflammatory and analgesic properties,[2] antihypertensive,[3] and hypolipidemic activities.[4] Recently, some studies have been focused on the anti-cancer features of hesperidin. Their findings have demonstrated that this biologically active compound can inhibit cell cycle progression and cell proliferation as well as promote apoptosis in different cancer cell lines (colon, breast, prostate, gastric, and pre-B cell acute lymphoblastic leukemia).[5],[6],[7],[8],[9] However, the mechanism by which hesperidin exhibits anticarcinogenic activity is not completely known. A body of evidence has proposed that flavonoids can inhibit carcinogenesis via down- or up-regulation of the fundamental element involved in various signaling pathways, regulating cell growth and proliferation, apoptosis, angiogenesis, migration, and metastasis, including the phosphoinositide 3–kinase (PI3K)/Akt.[10]

Phosphoinositide 3–kinase/Akt pathway contributes to the regulation of metabolism, cell survival, and proliferation in normal and malignant cells.[11],[12] Over-expression and over-activation of this signal transduction pathway result in the suppression of apoptosis and increase in the survival of tumor cells due to direct or indirect modulation of downstream regulatory molecules.[13],[14],[15]

Insulin is the main stimulator of PI3K/Akt signaling pathway.[16] Hyperinsulinemia is considered as a possible risk factor for cancer initiation and progression.[17] It is known that insulin stimulates the growth and proliferation of normal and malignant cells.[18] Insulin exerts its mitogenic effects through the recruitment of intracellular signaling cascades.[16]

It has been reported that PI3K/Akt pathway-dependent activation of nuclear factor-kappa B (NF-κB) plays a critical role in tumor progression, namely by inhibiting apoptosis.[19] We previously found that hesperidin induces apoptosis in human lymphoblastic NALM-6 cells (which possess wild-type p53 and constituent NF-κB expression [20]), via inhibition of NF-κB expression and activation.[9] The hypothesis of the present study was that hesperidin may inhibit NF-κB activation and trigger apoptosis in NALM-6 cells by inhibition of Akt activation. Therefore we investigated the effects of hesperidin on the inhibition of Akt activation and Akt-dependent NF-κB activation, in the presence of insulin (in hyperinsulinemia condition) as well as in the absence of insulin.

 > Materials and Methods Top

Reagents and antibodies

Hesperidin and 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (Sigma, St. Louis, Missouri, USA). Roswell Park Memorial Institute 1640 Medium and fetal bovine serum were purchased from Gibco (Life Technology, Grand Island, NY, USA). Wortmannin was obtained from Santa Cruz (Santa Cruz Biotechnology, CA, USA). All the primary antibodies for Western blot analysis, including anti-pAkt (ser473), anti-p glycogen synthase kinase-3 beta (GSK-3β) (ser9), anti-β-actin (C-2) (all rabbit monoclonal antibodies), anti-inhibitor of kappa B (IκB) kinase alpha (IKKα) (rabbit polyclonal antibody), and anti-p-IκBα (ser32/36) (mouse monoclonal antibody), were purchased from Cell Signaling (Cell Signaling Technology, MA, USA).

Cell viability assay

Cell viability was detected by MTT colorimetric assay.[21] Briefly, NALM-6 cells were placed at 5000/well in a 96-well culture plate. The cells were treated with hesperidin in the presence or absence of insulin for 24 and 48 h. After the medium removal, the cells were incubated with MTT solution (5 mg/ml in phosphate buffered saline) at 37°C for 4 h and then the formazan crystals were dissolved in 200 μL of dimethylsulfoxide. The absorbance of the samples was determined at 570 nm using a microplate reader (Bio-Tek, VA, USA).

Apoptotic death assay

Cell death detection ELISA Plus kit (Roche Applied Science, Mannheim, Germany) was used to evaluate apoptosis, following the manufacturer's instructions. Briefly, NALM-6 cells were seeded at 10,000/well in a 96-well round bottom microplate. The cells were incubated with the test compounds for 48 h. After the incubation, the cell lysates containing histone/DNA components were obtained and incubated in triplicate into the wells of the streptavidin-coated microplate. Subsequently, anti-histone-biotin antibody and anti-DNA-peroxidase antibody were added to well. Then, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as the peroxidase substrate, was added to the wells and generated the green end product. Finally, absorbance of the samples as an indicator of the extent of the histone/DNA fragments bounded to peroxidase was determined photometrically using a microplate reader at 405 nm (490 nm as a reference) (Bio-Tek, VA, USA).

Western blot analysis

The cells were lysed in the RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5 mM ethylenediaminetetraacetic acid, 10 mM NaF, 5 mM b-glycerophosphate, 0.1 mM Na3 VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 0.5% aprotinin). The protein concentrations were measured by the Bradford protein assay method (Bio-Rad, CA, USA). Same amounts of proteins were separated by a 10% SDS–polyacrylamide gel electrophoresis SDS and subsequently transferred to a polyvinylidene fluoride membrane (Roche Diagnostics, Mannheim, Germany) using a wet transfer system (Bio-Rad, Singapore). The proteins were detected using specific primary antibodies and the chemiluminescence detection system (ECL Prime, GE Healthcare Life Sciences, UK) according to the manufacturer's instruction.

Statistical analyses

Statistical Package for the Social Sciences (SPSS), version 20.00, (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses. The differences between the experimental variables were determined using one-way analysis of variance with Fisher's least significant difference test for multiple comparisons. P <0.05 was considered statistically significant.

 > Results Top

Hesperidin attenuated viability and induced apoptosis of NALM-6 cells in the absence or presence of insulin

To study the effect of insulin on cytostatic property of hesperidin in NALM-6 cells, these cells were treated with hesperidin in the presence and absence of insulin (100 nM) and then MTT assay was performed for 24 and 48 h. This concentration of insulin represents a hyperinsulinemic microenvironment same as serum insulin of obese subjects.[22] As shown in [Figure 1]a, hesperidin alone (25 and 50 μM) decreased the viability of NALM-6 cells after 24 h; however, the results were not significant compared to the untreated cells (P > 0.05). The inhibitory effect of hesperidin was significantly increased with the same concentrations after 48 h in a dose-dependent manner [Figure 1]b (P < 0.05). Insulin treatment (100 nM) slightly potentiated cell viability for 24 h; however, insulin incubation had no significant prosurvival effect on NALM-6 cells after 48 h. Hesperidin exerted its cytostatic effect even in the presence of insulin 48 after incubation. Wortmannin, a selective inhibitor of PI3K/Akt signaling pathway, was used as a positive control.[23] Wortmannin (0.5 μM) slightly decreased the viability of NALM-6 cells after 24 and 48 h [Figure 1]a and [Figure 1]b (P > 0.05).
Figure 1: Effect of hesperidin on cell viability and apoptosis in NALM-6 cells. (a and b) The NALM-6 cells were treated with hesperidin (25, 50 μM), insulin (100 nM), a combination of hesperidin and insulin, or wortmannin for 24 h (a) and 48 h (b) and cell viability was evaluated using 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All values are expressed as mean ± standard deviation (SD) of three independent experiments. *P < 0.05 versus control, **P < 0.05 versus insulin. (c) The apoptotic effect of hesperidin in NALM-6 cells was determined by cell death ELISA test after 48 h of the treatment by hesperidin (25 μM) alone or in the presence of insulin (100 nM), insulin (100 nM), or wortmannin (0.5 μM). +Control: positive control, a kit component containing DNA-histone complex. All the values are expressed as mean ± SD of two independent experiments. *P < 0.0001 versus control, **P < 0.0001 versus insulin

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According to marked effect of hesperidin after 48 h, apoptotic effect of hesperidin (minimum concentration, 25 μM) was analyzed by ELISA assay using the cell death detection ELISA Plus kit in the absence or presence of insulin. As shown in [Figure 1]c, hesperidin significantly promoted apoptosis in NALM-6 cells even in the presence of insulin after 48 h (P < 0.0001).

Hesperidin inhibits insulin-induced phosphorylation of Akt in NALM-6 cells

To investigate the role of hesperidin in the regulation of insulin-induced PI3K/Akt signaling activation, immunoblotting was performed as described in the materials and methods using antibody against phospho-Akt (Ser473) in NALM-6 cells. The cells were treated by hesperidin in the absence or presence of insulin. As shown in [Figure 2]a, insulin (100 nM) slightly elevated Akt phosphorylation 60 min after incubation; however, Akt phosphorylation was decreased at 120 min after incubation. Co-incubation of NALM-6 cells with insulin (100 nM) and hesperidin (25 and 50 μM) attenuated insulin-induced Akt phosphorylation up to 120 min after incubation.
Figure 2: Hesperidin reduced the insulin-induced phosphorylation of Akt and GSK-3β. (a) The NALM-6 cells were exposed to hesperidin (25 and 50 μM), insulin (100 nM), or hesperidin (25 and 50 μM) in the presence or absence of insulin (100 nM) and a group of cells was treated with wortmannin (0.5 μM) for 60 and 120 min. Then, protein extracts were provided. Western blot analysis was conducted using a specific antibody against Akt phosphorylated at Ser473 to evaluate the influence of hesperidin on the phosphorylation levels of Akt. WT: Wortmannin, Ins: Insulin; Ins + Hes: Insulin + Hesperidin; Hes: Hesperidin. (b) The cells were subjected to hesperidin (25 and 50 μM), insulin (100 nM), hesperidin (25 and 50 μM) with insulin (50 μM), or wortmannin (0.5 μM) for 90 min. Then, the cells were harvested, and the protein lysates were extracted to perform Western blot analysis using a specific antibody against GSK-3β phosphorylated at Ser9. Hes: Hesperidin; Ins + Hes: Insulin + Hesperidin

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Akt is constitutively active in most tumor cells in a PI3K-dependent or-independent manner.[24] As shown in [Figure 2]a, left panel, hesperidin had no significant inhibitory effect on non-insulin-dependent Akt phosphorylation 60 min after incubation; however, incubation of cells with 50 μM hesperidin showed a marked reduction in Akt phosphorylation at 120 min.

Glycogen synthase kinase-3 is a downstream substrate of PI3K/Akt signaling pathway.[25] To confirm the inhibitory effect of hesperidin on Akt activation, the effect of hesperidin on phosphorylation of GSK-3β at Akt phosphorylation site was assayed in NALM-6 cells. The cells were treated with two amounts of hesperidin (25 and 50 μM) in the presence or absence of 100 nM insulin. As shown in [Figure 2]b, hesperidin treatment decreased the insulin-induced phosphorylation of GSK-3β at Ser9 in NALM-6 cells.

Hesperidin prevents inhibitor of kappa B kinase alpha expression in NALM-6 cells

Inhibitor of kappa B kinases phosphorylation by upstream kinases is an important part of canonical NF-κB activation. Akt as a main upstream kinase, phosphorylates, and activates IKKs.[26],[27]

Our results indicated that hesperidin attenuated IKKα expression up to 240 min after treatment [Figure 3]a. It seems that hesperidin had a more potential role in reducing the total level of IKKα at the concentration of 25 μM up to 240 min after incubation. Incubation of NALM-6 cells with insulin (100 nM) decreased the total level of IKKα in comparison to control cells [Figure 3]b. In addition, co-treatment of cells with hesperidin (25 μM) and insulin (100 nM) reduced duration of hesperidin-induced IKKα down-regulation and IKKα expression was restarted 180 min after co-treatment [Figure 3]b.
Figure 3: (a and b) Hesperidin and insulin decreased total level of an inhibitor of kappa B kinase alpha (IKKα). The NALM-6 cells were treated with hesperidin (25 and 50 μM), insulin (100 nM), a combination of hesperidin (25 μM) and insulin (100 nM) for 45, 90, 180, and 240 min. Then, the cells were harvested and lysed, and the cell lysates were exposed to Western blot analysis using specific antibodies against IKKα

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Hesperidin reduces insulin-independent phosphorylation of inhibitor of kappa B

As shown in previous results, insulin decreased IKKα level in NALM-6 cells; therefore, it was plausible to presume that incubation of cells with insulin reduces the phosphorylation of IKKα main substrate, IκBα. As shown in [Figure 4], insulin had no significant effect on constitutive phosphorylation of IκBα, 60 min after incubation but IκBα phosphorylation increased at 120 min. Furthermore, hesperidin 25 μM had no inhibitory effect on phospho-IκBα in the absence or presence of insulin. Hesperidin at 50 μM concentration possessed a more potent inhibitory effect on IκBα phosphorylation in the absence or presence of insulin 120 min after treatment.
Figure 4: Hesperidin inhibited the phosphorylation of inhibitor of kappa B (IκB). The NALM-6 cells were incubated with hesperidin (25 and 50 μM), insulin (100 nM), hesperidin (25 and 50 μM) with insulin (100 nM), or wortmannin (0.5 μM) for 60 and 120 min. Then, the cell lysates were extracted and exposed to Western blot analysis using specific antibodies against p-IκB. Ins: Insulin; Ins + Hes: Insulin + Hesperidin; WT: Wortmannin; Hes: Hesperidin

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 > Discussion Top

Anti-cancer properties of flavonoids depend on different proposed mechanisms. Several lines of evidence suggested that flavonoids exert their cytotoxic and cytostatic effects through Akt inhibition.[28],[29] There is also robust evidence that suggests the role of NF-κB inhibition in apoptotic properties of flavonoids.[29],[30] It is well-recognized that Akt activation results in canonical NF-κB activation via direct phosphorylation of IKKs.[31],[32] Therefore, it is plausible to propose hesperidin as a potent NF-κB inhibitors which exerts its effect by inhibiting of Akt activation.

The results of the present study indicated that hesperidin induces cell death in NALM-6 cells as we previously showed.[9] Hesperidin promoted cell death even in the presence of insulin as a potent Akt activator in its signaling pathway.[16] We have previously shown that hesperidin suppresses NF-κB activation. Our present study showed that hesperidin may exert this effect by suppression of IKKα expression and decrease in IκB phosphorylation in an Akt-dependent manner.

Phosphoinositide 3–kinase/Akt over-activation has been frequently observed in a large number of human cancers, such as glioma (almost 55%), thyroid carcinoma (80–100%), gastric carcinoma (almost 80%), and pancreatic carcinoma (30–70%).[33] Mutations in components of PI3K/Akt pathway have also been detected in most malignancies, specifically in hematological malignancies.[34] Akt activation has been shown in almost 70% of acute myeloid leukemia.[33] Human pre-B acute lymphoblastic leukemia cell lines, NALM-6 and Reh cells carry epigenetically inactivate PTEN that causes hyperactivation of Akt in this cells.[35],[36] The results of the present study demonstrated that the incubation of NALM-6 cells with hesperidin inhibited the insulin-induced activation of Akt in these cells while just the high concentration of hesperidin could decrease the constitutive activation of Akt. It seems that insulin exerts its metabolic effects through the activation of Akt2[37] and Akt2 function is more specific for the insulin receptor signaling pathway.[38] Due to the stimulation of cell with insulin, Akt1 and Akt2 are translocated to the cell membrane and activated by phosphorylation, but Akt2 is accumulated in cell membrane more than Akt1 in response to insulin.[39] Akt2 over-expression has been observed in ovarian and pancreatic cancers.[40] Furthermore, insulin-induced activation of Akt2 has been reported in human ovarian carcinoma cells.[41] Furthermore, we found that hesperidin inhibited insulin-induced GSK-3β phosphorylation in NALM-6 cells. Since GSK-3β is a downstream target of Akt2,[41] these results may suggest that hesperidin more potently exerts its anti-cancer effects by suppressing of Akt2 phosphorylation.

Nuclear factor-kappa B has also been proposed as a survival factor in tumor cells specifically in hematological malignancies.[42] NF-κB stimulation increases resistance to apoptosis and promotes cell proliferation.[8] Several research has been shown that flavonoids can block NF-κB signaling. For instance, it has been shown that quercetin triggers apoptosis in human salivary adenoid cystic carcinoma through down-regulation of PI3K/Akt/IKKα and subsequent suppression of NF-κB activation.[29] In addition, fisetin stimulates cell cycle arrest and apoptosis through inhibition of NF-κB signal in bladder cancer cells.[43] We observed that hesperidin decreased total level of IKK. Since hesperidin (high concentration) was determined to reduce the phosphorylation of Akt and IκB, it seems that this flavonoid inhibits IKK expression and results in a reduction in phosphorylation of IκB and subsequent inhibition of NF-κB activation. We also indicated that insulin acutely suppresses IKKα level in NALM-6 cells in correlation with increase in phosphorylation of IκB. These findings suggest that the reduction in total form of IKK can be the result of increase in the phosphorylated form of IKK, due to stimulation by insulin. Hence, insulin might induce NF-κB activation by stimulating Akt/IKK pathway. In conclusion, our study indicates that hesperidin has cytotoxic and proapoptotic activities by targeting Akt and NF-κB pathways, which can sensitize cancer cells to chemotherapeutic agents. These results reveal that hesperidin may serve as an adjunct to chemotherapy in the treatment of human cancers, which are related or unrelated to hyperinsulinemia.

 > Acknowledgments Top

This article is based on a thesis and supported by Master research grants (P/25/47/1023) from National Institute of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

 > References Top

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


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