- Email: [email protected]
Akt pathway activation
Chinese Journal of Natural Medicines 2014, 12(1): 0015−0019
Chinese Journal of Natural Medicines
Ursolic acid induces U937 cells differentiation by PI3K/Akt pathway activation DENG Lin 1, 2, ZHANG Rui 1, 2, TANG Feng 1, 2, LI Chen 1, 2, XING Ying-Ying 1, 2, XI Tao 1, 2* 1 2
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China; Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China Available online 20 Jan. 2014 [ABSTRACT] AIM: Ursolic acid (UA), a pentacyclic triterpenoid, is used as an anti-inflammatory and anti-cancer agent. There were few studies on the effects of UA on differentiation, and this is the first time to elucidate the potential effect and molecular mechanism of UA on inducing differentiation in the human leukemia cell line U937. METHODS: Wright-Giemsa staining, nitroblue tetrazolium reduction assay and flow cytometric analysis were utilized to demonstrate the differentiation of U937 cells induced by UA. Western blotting and immunofluorescence assay were used to investigate the possible mechanism. RESULTS: It was found that UA could induce the differentiation of U937cells and Akt-activity was significantly increased during differentiation. Additionally, LY294002, a PI3K-Akt inhibitor, could block the differentiation of U937 cells induced by UA. CONCLUSION: UA could induce the differentiation of U937 cells by activating the PI3K/Akt pathway, and it could be a potential candidate as a differentiation-inducing agent for the therapy of leukemia.
[KEY WORDS] Ursolic acid (UA); U937; Differentiation; PI3K/Akt ; Phosphorylation
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)01-0015-05
Introduction Cancer, including solid tumors and leukemias, is a primary threat to the health of human beings. Acute myeloid leukemia (AML) is caused by a blockade in differentiation that leads to an accumulation of abnormal cells. Therefore, the induction of AML cell differentiation is becoming an ideal therapeutic method [1]. In 1986, great breakthroughs in differentiation therapy with all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia were achieved in Shanghai [2]. However, developing drug resistance and the fetal “ATRA syndrome” limited its clinical use [3]. It is reported that there are cardiac stunning during ATRA-induced differentiation in acute promyelocytic leukemia [4]. Thus, in recent years efforts have been made to develop other effective differentiation inducers with low side effects, and to investigate their mechanism of action. [Received on] 08-May-2012 [*Corresponding author] XI Tao: Prof., Tel/Fax: 86-25-83271022; E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
Ursolic acid (UA), a pentacyclic triterpene acid, is widely distributed in foods and plants. Many studies reported that UA could inhibit proliferation and induce apoptosis in many cancer cell lines, such as those of leukemia, lung cancer, and endometrial cancer [5-10]. Moreover, early studies concerning the anticancer effects of UA mainly focused on cell cycle regulation and cell apoptosis induction, whereas studies on differentiation induction were rare. The serine/threonine protein kinase B/Akt (PKB) is a downstream effector of the phosphoinositide 3-kinase (PI3K) and acts as an essential modulator in many processes, including proliferation, differentiation, and cell survival [11-12]. In normal cells, PKB/Akt is found in the cytosol in a catalytically inactive state. Activation of PI3K by irritants leads to generation of phosphatidylinositol (3, 4, 5)-trisphosphate (PtdIns 3, 4, 5 P3)2 which recruits inactive PKB/Akt to the plasma membrane [13]. The complete activation of PKB/Akt requires both the phosphorylation of Thr308 by phosphoinositide-dependent protein kinase-1 (PDK1) and of Ser473 in the hydrophobic motif [14-15]. Once activated, PKB/Akt regulates the activities of downstream targets in the cytoplasm and nucleus. In this study, the effects of UA on the differentiation of human leukemia cell line U937 were investigated, and the
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associated mechanism studied. The data showed for the first time that UA could induce differentiation of U937 cells through activating the PI3K/Akt signaling pathway.
Materials and Methods Reagents UA (purity > 98%) was dissolved in dimethyl sulfoxide (DMSO; Sigma, USA) and stored at −20 °C. Nitroblue tetrazolium (NBT), TPA and ATRA, were purchased from Sigma (Sigma, St. Louis, MO, USA). Rabbit antiphospho-Akt (Ser473), rabbit antiphospho-Akt (Thr308), rabbit anti-Akt, rabbit antiphospho-PDK1 (Ser241) and LY204002 were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell culture U937 human leukemia cells were cultured in RPMI 1640 medium (Gibco/BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. Cultures were replaced every two days to maintain a log-phase growth of the cells. Differentiation assay (i) NBT reduction assay. The NBT reducing activity was determined by the method described previously [16]. In brief, after treatment with 30 μmol·L−1 of UA, 1 × 106 cells were harvested, incubated in a serum-free medium containing 2 mg·mL−1 NBT and 1 mg·mL−1 TPA for 30 min at 37 °C, then washed with 70% methanol, and finally lysed in 500 mL of 2 mol·L−1 KOH overnight. The nitroblue diformazan deposit was dissolved in 600 μL/sample of DMSO, and the absorbance was measured at 570 nm. (ii) Analysis of morphological changes. The cells were collected, fixed with methanol on the slides and stained with the Wright–Giemsa solution, washed with deionized water, air-dried, and then observed under the light microscope (YS100; Nikon, Japan). (iii) Flow cytometric analysis. The cells were washed twice with the washing buffer (phosphate buffered saline containing 0.1% sodium azide, and 0.2% (W/V) bovine serum albumin), and then incubated in ice water with the anti-CD11b antibody conjugated with phycoerythrin and the anti-CD14 antibody (ebioscience, San Diego, CA, USA) conjugated with fluorescein isothiocyanate for 30 min. After washing, the cells were resuspended in 500 mL of wash buffer, and finally analyzed by FACSalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Western blot analysis Proteins from cell lysate and nuclear fraction were harvested. Protein concentrations were determined by Bradford protein assay (Bio-Rad). Equal amounts of proteins from cell lysate (100–200 μg) or nuclear fraction (70–100 μg) were separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically onto polyvinylidene difluoride membranes. Polyclonal rabbit antibodies against Akt, phospho-Akt (Ser 473), phospho-Akt (Thr308), and phospho-PDK1 (Ser241) were purchased from Cell Signaling Technology (Beverly, MA, USA). Proteins were visualized with an enhanced chemilumi-
nescence detection system (Amersham, UK). Immunofluorescence microscopy Collected U937 cells were washed gently with PBS, and fixed in methanol at −20 °C. Then the cells were permeabilized with 0.05% Triton X-100 in PBS at −4 °C for 30 min and washed three times in PBS. After being washed, the cells were incubated in a blocking buffer (3% BSA) for 60 min at RT. The cells were then incubated with the primary antibodies (rabbit anti-Akt) overnight at 4 °C. After another washing with PBS, the slides were incubated with the secondary antibody, namely FITC-conjugated goat anti-rabbit immunoglobulin G, in PBS for 60 min at RT. Nuclei were stained with DAPI for 30 min at RT. Standard epifluorescence was observed and photographed with a confocal laser scanning microscope.
Results UA induces U937 leukemia cell lines differentiation Wright–Giemsa staining was used to detect the morphological changes of U937 cells after treatment with UA. The negative group treated with 0.3% DMSO was undifferentiated, with round, regular cell margins, and large nuclei. By contrast, after treatment with UA (30 μmol·L−1) for 4 days, condensed nuclei, and abundant cytoplasm and vacuoles were observed in the cells, suggesting typical differentiation characteristics of U937 cells (Fig. 1A). Subsequently, the NBT reduction assay was performed using UA（30 μmol·L−1）and ATRA (1 μmol·L−1) as a positive control. Four days after treatment, a significant increase in NBT reduction ability was observed (Fig. 1B). The morphological changes of U937 cells after UA treatment preliminarily suggested monocytic differentiation of the cells. Then the cells were labeled with CD14 and CD11b, which are up-regulated during myelomonocytic differentiation [17-18] . The results further confirmed the differentiation-inducing effect of UA. The expressions of CD14 and CD11b were significantly increased after UA and ATRA treatment compared with the negative control, suggesting that UA could induce the monocytic differentiation of U937 cells (Fig. 1C). UA induced phosphorylation of Akt and its nuclear translocation in U937 cells The results showed that Akt was phosphorylated at Thr308 and Ser473 in U937 cells, and this phosphorylation was significantly increased during 24 to 96 h after treatment with UA (Fig. 2A, 2B). In isolated nuclei, the levels of Ser 473 and Thr308 p-Akt were increased after treatment with UA and ATRA (Fig. 2C). The complete activation of PKB/Akt requires both the phosphorylation of Thr308 by phosphoinositide-dependent protein kinase-1 (PDK1) and of Ser473 in the hydrophobic motif [19]. The phosphorylation of PDK1 was determined and it was found that PDK1 was phosphorylated at Ser241 in U937 cells after UA treatment (Fig. 2A).
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Fig. 1 Effects of ursolic acid (UA) on the differentiation of U937 cells. (A) UA induces morphologic changes of U937 cells. (B) Nitroblue tetrazolium (NBT) reduction assay after UA and LY294002 treatment. Data were means ± SEM, n = 3, *P < 0.05, **p < 0.01. (C) Flow cytometric analysis to examine the increase of the differentiation marker after treatment with UA (30 μmol·L−1), ATRA (1 μmol·L−1), and LY294002 (10 μmol·L−1)
To further demonstrate that Akt was activated in the nuclei of UA-treated cells, immunofluorescence microscopy was used to observe the nuclear translocation of active Akt. As shown in Fig. 2D, Akt was located mainly in the cytoplasm before UA treatment, and accumulated in the nucleus 96 h after treatment with UA. LY294002 inhibits U937 leukemia cell lines differentiation To further demonstrate whether UA induced the differentiation of U937 cells by the activation of the PI3K/Akt pathway, the PI3 kinase inhibitor LY294002 was used to study the expression of cell surface differentiation markers. When the U937 cells were treated with LY294002 (10 μmol·L−1) for 30 min before the addition of UA (30 μmol·L−1) for further incubation for 4 days, phosphorylation of Akt (Fig. 3) and the nuclear translocation of active Akt (Fig. 2D ) were inhibited. Furthermore, there was no increase in NBT reduction ability and the expression of CD14 and CD11b was also blocked after treatment with LY294002 (Fig. 1B, 1C). These results further confirmed that UA induced the differentiation of U937cells through activating the PI3K/Akt pathway.
Discussion Leukemia is a serious threat to the health of people. In 1986, differentiation therapy with ATRA had made a great breakthrough in the area of leukemia therapy, but drug resistance limited its use. As a result, more efforts are needed to develop alternative and effective differentiation inducers in recent years. The study of the molecular mechanisms involved is also of great importance.
In this study, it was shown that ursolic acid (UA) could induce the differentiation of U937 cells, and the expression of the specific monocytic surface markers CD14 and CD11b significantly increased. Moreover, NBT reduction capability also increased after UA treatment. Though the results showed the differentiation-inducing effect of ATRA is more efficient than that of UA, UA is less toxic than ATRA for ATRA could cause the fetal “ATRA syndrome”, a severe side effect, and the development of drug-resistance. Furthermore, earlier studies about the cytotoxicity of UA have shown that UA is less harmful to normal cells, such as the serum-free mouse embryo cell line and the widely cultured normal mouse hepatocytes [20-21]. Many studies have reported that the PI3K/Akt signaling pathway plays an important role in the monocytic and granulocytic differentiation of different leukemic cell lines induced by various differentiating agents, including ATRA and EPO. Akt/PKB was phosphorylated and potently activated when the differentiation-inducing factor-1 (DIF-1) induced differentiation in K562 cells [22]. Ishida et al. [23] reported the role of the PI3K/Akt pathway in the differentiation of HL60 cells induced by ATRA. Matkovic et al. [13] reported that the expression of p-Akt, especially Akt, in the nuclei, significantly increased during the differentiation of HL60 cells induced by ATRA. In addition, Wang et al. [24] found that Akt could regulate the differentiation of lymphocytes induced by VD3 through the Raf/MEK/ERK MAPK signaling pathway. There are also studies about the nuclear translocation of Akt during the differentiation of K562 cells induced by EPO [25].
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Fig. 2 Effects of ursolic acid (UA) on the activation of PI3K/Akt pathway in U937 cells. (A) Western blotting of phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt and phospho-PDK (Ser241) from the total proteins in the cells after treatment with UA (30 μmol·L−1) for 24, 48, 72 and 96 h. (B) Analysis of phosphorylated Akt in U937 cells after treatment with different concentrations of UA for 96 h. (C) Western blotting of phospho-Akt (Ser473) and phospho-Akt (Thr308) of nuclei protein 96 h after treatment with UA (30 μmol·L−1). (D) Analysis of Akt distribution in U937 cells treated with UA and LY294002 by fluorescence microscopy. Akt protein was labeled by fluorescein isothiocyanate (FITC, green) and the nucleus was labeled by DAPI (blue)
Fig. 3 Effects of LY294002 on activation of the PI3K/Akt pathway in U937 cells. Western blotting of phospho-Akt (Ser473), phospho-Akt (Thr308), and total Akt in U937 cells treated with LY294002 (10 μmol·L−1) or untreated
However, the effects of UA on the PI3K/Akt signaling pathway during the differentiation of U937 cells have not been reported. In this study, it was found that UA could induce the differentiation of U937 cells through activation of the PI3K/Akt signaling pathway, and modulate the levels of phosphor-Akt in a time-dependant manner. To further support the hypothesis, a specific PI3K inhibitor LY294002 was utilized in this study, and it was found that UA-induced differentiation was blocked by LY294002, suggesting that the activation of Akt is essential for UA-induced differentiation. In summary, the role of UA in inducing monocytic differentiation of U937 cells by the activation of PI3K/Akt signaling pathway has been found for the first time in this study.
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Studies on the mechanisms of UA-induced differentiation could benefit the development of new pharmacological agents with potential therapeutic value and fewer side effects for the treatment of leukemia.
Acknowledgements The authors thank Prof. CHEN Wu and Dr. LU Yun-Hua, College of CHUN Yi, for the gift of highly purified UA. The authors also thank Prof. GUO Qing-Long for providing the U937 cell line used in this study, DENG Xue-Peng for helpful comments on the manuscript, and the members of the XI Tao lab for supportive discussion.
References [1]
Nowak D, Stewart D, Koeffler HP. Differentiation therapy of leukemia: 3 decades of development[J]. Blood, 2009, 113(16): 3655-3665. [2] Pierce JN, Stein S. Multiple diversity with onindependent fading[J]. Proc IRE, 1960, 48(1): 89-104. [3] Warrell RP Jr. Retinoid resistance in acute promyelocytic leukemia: new mechanisms, strategies, and implications[J]. Blood, 1993, 82: 1949-1953. [4] De Santis GC, Madeira MIA, de Oliveira LCO, et al. Cardiac stunning as a manifestation of ATRA differentiation syndrome in acute promyelocytic leukemia[J]. Med Oncol, 2012, 29: 248-250. [5] Tohda S, Curtis JE, McCulloch EA, et al. Comparison of the effects of all-trans and cis-retinoic acid on the blast stem cells of acute myeloblastic leukemia in culture[J]. Leukemia, 1992, 6(7): 656-661. [6] Choi BM, Park R, Pae HO, et al. Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells[J]. Pharmacol Toxicol, 2000, 86(2): 53–58. [7] Kassi E, Papoutsi Z, Pratsinis H, et al. Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells[J]. Cancer Res Clin Oncol, 2007, 133: 493–500. [8] Hsu YL, Kuo PL, Lin CC: Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells[J]. Life Sci, 2004, 75(19): 2303–2316. [9] Achiwa Y, Hasegawa K, Komiya T, et al. Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer SNG-II cells[J]. Oncol Rep, 2005, 13(1): 51-57. [10] Manu KA, Kuttan G. Ursolic acid induces apoptosis by activating p53 and caspase-3 gene expressions and suppressing NF-kappa B mediated activation of bcl-2 in B16F-10 melanoma cells[J]. Int Immunopharmacol, 2008, 8(7): 974-981. [11] Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts[J]. Genes Dev, 1999, 13(22): 2905-2927.
[12] Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004, 1697(1-2): 3-16. [13] Matkovic K, Brugnoli F, Bertagnolo V, et al. The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells[J]. Leukemia, 2006, 20: 941-951. [14] Williams MR, Arthur JS, Balendran A, et al. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells[J]. Curr Biol, 2000, 10(8): 439-448. [15] Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[J]. Science, 2005, 307(5712): 1098-1101. [16] Poon KH, Zhang J, Wang C, et al. Betulinic acid enhances 1α, 25-dihydroxyvitamin D3-induced differentiation in human HL-60 promyelocytic leukemia cells[J]. Anticancer Drugs, 2004, 15(6): 619-624. [17] Brackman D, Lund F, Aarskog D. Expression of leukocyte differentiation antigens during the differentiation of HL-60 cells induced by 1, 25-dihydroxyvitamin D3: comparison with the maturation of normal monocytic and granulocytic bone marrow cells[J]. Leukoc Biol, 1995, 58(5): 547-555. [18] Hmama Z, Nandan D, Sly L, et al. 1alpha,25- dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex[J]. Exp Med, 1999, 190(11): 1583-1594. [19] Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1[J]. EMBO, 1996, 15(23): 6541-6551. [20] Yamaguchi H, Noshita T, Kidachi Y, et al. Isolation of ursolic acid from apple peels and its specific efficacy as a potent antitumor agent[J]. Health Sci, 2008, 54: 654-660. [21] Tian Z, Lin G, Zheng RX, et al. Anti-hepatoma activity and mechanism of ursolic acid and its derivatives isolated from Aralia decaisneana[J]. World J Gastroenterol, 2006, 12(6): 874-879. [22] Ishida S, Shigemoto-Mogami Y, Shinozaki Y, et al. Differential modulation of PI3- kinase/Akt pathway during all-trans retinoic acid-and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis[J]. Biochem Pharmacol, 2004, 68(11): 2177- 2186. [23]Kubohara Y, Hosaka K. The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells[J]. BBRC, 1999, 263(3): 790-796. [24] Wang J, Zhao Y, Kauss MA, et al. Akt regulates vitamin D3-induced leukemia cell functional differentiation via Raf/MEK/ERK MAPK signaling[J]. Eur J Cell Biol, 2009, 88(2): 103-115. [25] Missiroli S, Etro D, Buontempo F, et al. Nuclear translocation of active AKT is required for erythroid differentiation in erythropoietin-treated K562 erythroleukemia cells[J]. Int J Biochem Cell Biol, 2009, 41(3): 570-577.
Cite this article as: DENG Lin, ZHANG Rui, TANG Feng, LI Chen, XING Ying-Ying, XI Tao. Ursolic acid induces U937 cells differentiation by PI3K/Akt pathway activation [J]. Chinese Journal of Natural Medicines, 2014, 12(1): 15-19
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Chinese Journal of Natural Medicines
Ursolic acid induces U937 cells differentiation by PI3K/Akt pathway activation DENG Lin 1, 2, ZHANG Rui 1, 2, TANG Feng 1, 2, LI Chen 1, 2, XING Ying-Ying 1, 2, XI Tao 1, 2* 1 2
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China; Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China Available online 20 Jan. 2014 [ABSTRACT] AIM: Ursolic acid (UA), a pentacyclic triterpenoid, is used as an anti-inflammatory and anti-cancer agent. There were few studies on the effects of UA on differentiation, and this is the first time to elucidate the potential effect and molecular mechanism of UA on inducing differentiation in the human leukemia cell line U937. METHODS: Wright-Giemsa staining, nitroblue tetrazolium reduction assay and flow cytometric analysis were utilized to demonstrate the differentiation of U937 cells induced by UA. Western blotting and immunofluorescence assay were used to investigate the possible mechanism. RESULTS: It was found that UA could induce the differentiation of U937cells and Akt-activity was significantly increased during differentiation. Additionally, LY294002, a PI3K-Akt inhibitor, could block the differentiation of U937 cells induced by UA. CONCLUSION: UA could induce the differentiation of U937 cells by activating the PI3K/Akt pathway, and it could be a potential candidate as a differentiation-inducing agent for the therapy of leukemia.
[KEY WORDS] Ursolic acid (UA); U937; Differentiation; PI3K/Akt ; Phosphorylation
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)01-0015-05
Introduction Cancer, including solid tumors and leukemias, is a primary threat to the health of human beings. Acute myeloid leukemia (AML) is caused by a blockade in differentiation that leads to an accumulation of abnormal cells. Therefore, the induction of AML cell differentiation is becoming an ideal therapeutic method [1]. In 1986, great breakthroughs in differentiation therapy with all-trans-retinoic acid (ATRA) in acute promyelocytic leukemia were achieved in Shanghai [2]. However, developing drug resistance and the fetal “ATRA syndrome” limited its clinical use [3]. It is reported that there are cardiac stunning during ATRA-induced differentiation in acute promyelocytic leukemia [4]. Thus, in recent years efforts have been made to develop other effective differentiation inducers with low side effects, and to investigate their mechanism of action. [Received on] 08-May-2012 [*Corresponding author] XI Tao: Prof., Tel/Fax: 86-25-83271022; E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
Ursolic acid (UA), a pentacyclic triterpene acid, is widely distributed in foods and plants. Many studies reported that UA could inhibit proliferation and induce apoptosis in many cancer cell lines, such as those of leukemia, lung cancer, and endometrial cancer [5-10]. Moreover, early studies concerning the anticancer effects of UA mainly focused on cell cycle regulation and cell apoptosis induction, whereas studies on differentiation induction were rare. The serine/threonine protein kinase B/Akt (PKB) is a downstream effector of the phosphoinositide 3-kinase (PI3K) and acts as an essential modulator in many processes, including proliferation, differentiation, and cell survival [11-12]. In normal cells, PKB/Akt is found in the cytosol in a catalytically inactive state. Activation of PI3K by irritants leads to generation of phosphatidylinositol (3, 4, 5)-trisphosphate (PtdIns 3, 4, 5 P3)2 which recruits inactive PKB/Akt to the plasma membrane [13]. The complete activation of PKB/Akt requires both the phosphorylation of Thr308 by phosphoinositide-dependent protein kinase-1 (PDK1) and of Ser473 in the hydrophobic motif [14-15]. Once activated, PKB/Akt regulates the activities of downstream targets in the cytoplasm and nucleus. In this study, the effects of UA on the differentiation of human leukemia cell line U937 were investigated, and the
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DENG Lin, et al. / Chin J Nat Med, 2014, 12(1): 15−19
associated mechanism studied. The data showed for the first time that UA could induce differentiation of U937 cells through activating the PI3K/Akt signaling pathway.
Materials and Methods Reagents UA (purity > 98%) was dissolved in dimethyl sulfoxide (DMSO; Sigma, USA) and stored at −20 °C. Nitroblue tetrazolium (NBT), TPA and ATRA, were purchased from Sigma (Sigma, St. Louis, MO, USA). Rabbit antiphospho-Akt (Ser473), rabbit antiphospho-Akt (Thr308), rabbit anti-Akt, rabbit antiphospho-PDK1 (Ser241) and LY204002 were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell culture U937 human leukemia cells were cultured in RPMI 1640 medium (Gibco/BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. Cultures were replaced every two days to maintain a log-phase growth of the cells. Differentiation assay (i) NBT reduction assay. The NBT reducing activity was determined by the method described previously [16]. In brief, after treatment with 30 μmol·L−1 of UA, 1 × 106 cells were harvested, incubated in a serum-free medium containing 2 mg·mL−1 NBT and 1 mg·mL−1 TPA for 30 min at 37 °C, then washed with 70% methanol, and finally lysed in 500 mL of 2 mol·L−1 KOH overnight. The nitroblue diformazan deposit was dissolved in 600 μL/sample of DMSO, and the absorbance was measured at 570 nm. (ii) Analysis of morphological changes. The cells were collected, fixed with methanol on the slides and stained with the Wright–Giemsa solution, washed with deionized water, air-dried, and then observed under the light microscope (YS100; Nikon, Japan). (iii) Flow cytometric analysis. The cells were washed twice with the washing buffer (phosphate buffered saline containing 0.1% sodium azide, and 0.2% (W/V) bovine serum albumin), and then incubated in ice water with the anti-CD11b antibody conjugated with phycoerythrin and the anti-CD14 antibody (ebioscience, San Diego, CA, USA) conjugated with fluorescein isothiocyanate for 30 min. After washing, the cells were resuspended in 500 mL of wash buffer, and finally analyzed by FACSalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Western blot analysis Proteins from cell lysate and nuclear fraction were harvested. Protein concentrations were determined by Bradford protein assay (Bio-Rad). Equal amounts of proteins from cell lysate (100–200 μg) or nuclear fraction (70–100 μg) were separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically onto polyvinylidene difluoride membranes. Polyclonal rabbit antibodies against Akt, phospho-Akt (Ser 473), phospho-Akt (Thr308), and phospho-PDK1 (Ser241) were purchased from Cell Signaling Technology (Beverly, MA, USA). Proteins were visualized with an enhanced chemilumi-
nescence detection system (Amersham, UK). Immunofluorescence microscopy Collected U937 cells were washed gently with PBS, and fixed in methanol at −20 °C. Then the cells were permeabilized with 0.05% Triton X-100 in PBS at −4 °C for 30 min and washed three times in PBS. After being washed, the cells were incubated in a blocking buffer (3% BSA) for 60 min at RT. The cells were then incubated with the primary antibodies (rabbit anti-Akt) overnight at 4 °C. After another washing with PBS, the slides were incubated with the secondary antibody, namely FITC-conjugated goat anti-rabbit immunoglobulin G, in PBS for 60 min at RT. Nuclei were stained with DAPI for 30 min at RT. Standard epifluorescence was observed and photographed with a confocal laser scanning microscope.
Results UA induces U937 leukemia cell lines differentiation Wright–Giemsa staining was used to detect the morphological changes of U937 cells after treatment with UA. The negative group treated with 0.3% DMSO was undifferentiated, with round, regular cell margins, and large nuclei. By contrast, after treatment with UA (30 μmol·L−1) for 4 days, condensed nuclei, and abundant cytoplasm and vacuoles were observed in the cells, suggesting typical differentiation characteristics of U937 cells (Fig. 1A). Subsequently, the NBT reduction assay was performed using UA（30 μmol·L−1）and ATRA (1 μmol·L−1) as a positive control. Four days after treatment, a significant increase in NBT reduction ability was observed (Fig. 1B). The morphological changes of U937 cells after UA treatment preliminarily suggested monocytic differentiation of the cells. Then the cells were labeled with CD14 and CD11b, which are up-regulated during myelomonocytic differentiation [17-18] . The results further confirmed the differentiation-inducing effect of UA. The expressions of CD14 and CD11b were significantly increased after UA and ATRA treatment compared with the negative control, suggesting that UA could induce the monocytic differentiation of U937 cells (Fig. 1C). UA induced phosphorylation of Akt and its nuclear translocation in U937 cells The results showed that Akt was phosphorylated at Thr308 and Ser473 in U937 cells, and this phosphorylation was significantly increased during 24 to 96 h after treatment with UA (Fig. 2A, 2B). In isolated nuclei, the levels of Ser 473 and Thr308 p-Akt were increased after treatment with UA and ATRA (Fig. 2C). The complete activation of PKB/Akt requires both the phosphorylation of Thr308 by phosphoinositide-dependent protein kinase-1 (PDK1) and of Ser473 in the hydrophobic motif [19]. The phosphorylation of PDK1 was determined and it was found that PDK1 was phosphorylated at Ser241 in U937 cells after UA treatment (Fig. 2A).
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Fig. 1 Effects of ursolic acid (UA) on the differentiation of U937 cells. (A) UA induces morphologic changes of U937 cells. (B) Nitroblue tetrazolium (NBT) reduction assay after UA and LY294002 treatment. Data were means ± SEM, n = 3, *P < 0.05, **p < 0.01. (C) Flow cytometric analysis to examine the increase of the differentiation marker after treatment with UA (30 μmol·L−1), ATRA (1 μmol·L−1), and LY294002 (10 μmol·L−1)
To further demonstrate that Akt was activated in the nuclei of UA-treated cells, immunofluorescence microscopy was used to observe the nuclear translocation of active Akt. As shown in Fig. 2D, Akt was located mainly in the cytoplasm before UA treatment, and accumulated in the nucleus 96 h after treatment with UA. LY294002 inhibits U937 leukemia cell lines differentiation To further demonstrate whether UA induced the differentiation of U937 cells by the activation of the PI3K/Akt pathway, the PI3 kinase inhibitor LY294002 was used to study the expression of cell surface differentiation markers. When the U937 cells were treated with LY294002 (10 μmol·L−1) for 30 min before the addition of UA (30 μmol·L−1) for further incubation for 4 days, phosphorylation of Akt (Fig. 3) and the nuclear translocation of active Akt (Fig. 2D ) were inhibited. Furthermore, there was no increase in NBT reduction ability and the expression of CD14 and CD11b was also blocked after treatment with LY294002 (Fig. 1B, 1C). These results further confirmed that UA induced the differentiation of U937cells through activating the PI3K/Akt pathway.
Discussion Leukemia is a serious threat to the health of people. In 1986, differentiation therapy with ATRA had made a great breakthrough in the area of leukemia therapy, but drug resistance limited its use. As a result, more efforts are needed to develop alternative and effective differentiation inducers in recent years. The study of the molecular mechanisms involved is also of great importance.
In this study, it was shown that ursolic acid (UA) could induce the differentiation of U937 cells, and the expression of the specific monocytic surface markers CD14 and CD11b significantly increased. Moreover, NBT reduction capability also increased after UA treatment. Though the results showed the differentiation-inducing effect of ATRA is more efficient than that of UA, UA is less toxic than ATRA for ATRA could cause the fetal “ATRA syndrome”, a severe side effect, and the development of drug-resistance. Furthermore, earlier studies about the cytotoxicity of UA have shown that UA is less harmful to normal cells, such as the serum-free mouse embryo cell line and the widely cultured normal mouse hepatocytes [20-21]. Many studies have reported that the PI3K/Akt signaling pathway plays an important role in the monocytic and granulocytic differentiation of different leukemic cell lines induced by various differentiating agents, including ATRA and EPO. Akt/PKB was phosphorylated and potently activated when the differentiation-inducing factor-1 (DIF-1) induced differentiation in K562 cells [22]. Ishida et al. [23] reported the role of the PI3K/Akt pathway in the differentiation of HL60 cells induced by ATRA. Matkovic et al. [13] reported that the expression of p-Akt, especially Akt, in the nuclei, significantly increased during the differentiation of HL60 cells induced by ATRA. In addition, Wang et al. [24] found that Akt could regulate the differentiation of lymphocytes induced by VD3 through the Raf/MEK/ERK MAPK signaling pathway. There are also studies about the nuclear translocation of Akt during the differentiation of K562 cells induced by EPO [25].
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Fig. 2 Effects of ursolic acid (UA) on the activation of PI3K/Akt pathway in U937 cells. (A) Western blotting of phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt and phospho-PDK (Ser241) from the total proteins in the cells after treatment with UA (30 μmol·L−1) for 24, 48, 72 and 96 h. (B) Analysis of phosphorylated Akt in U937 cells after treatment with different concentrations of UA for 96 h. (C) Western blotting of phospho-Akt (Ser473) and phospho-Akt (Thr308) of nuclei protein 96 h after treatment with UA (30 μmol·L−1). (D) Analysis of Akt distribution in U937 cells treated with UA and LY294002 by fluorescence microscopy. Akt protein was labeled by fluorescein isothiocyanate (FITC, green) and the nucleus was labeled by DAPI (blue)
Fig. 3 Effects of LY294002 on activation of the PI3K/Akt pathway in U937 cells. Western blotting of phospho-Akt (Ser473), phospho-Akt (Thr308), and total Akt in U937 cells treated with LY294002 (10 μmol·L−1) or untreated
However, the effects of UA on the PI3K/Akt signaling pathway during the differentiation of U937 cells have not been reported. In this study, it was found that UA could induce the differentiation of U937 cells through activation of the PI3K/Akt signaling pathway, and modulate the levels of phosphor-Akt in a time-dependant manner. To further support the hypothesis, a specific PI3K inhibitor LY294002 was utilized in this study, and it was found that UA-induced differentiation was blocked by LY294002, suggesting that the activation of Akt is essential for UA-induced differentiation. In summary, the role of UA in inducing monocytic differentiation of U937 cells by the activation of PI3K/Akt signaling pathway has been found for the first time in this study.
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Studies on the mechanisms of UA-induced differentiation could benefit the development of new pharmacological agents with potential therapeutic value and fewer side effects for the treatment of leukemia.
Acknowledgements The authors thank Prof. CHEN Wu and Dr. LU Yun-Hua, College of CHUN Yi, for the gift of highly purified UA. The authors also thank Prof. GUO Qing-Long for providing the U937 cell line used in this study, DENG Xue-Peng for helpful comments on the manuscript, and the members of the XI Tao lab for supportive discussion.
References [1]
Nowak D, Stewart D, Koeffler HP. Differentiation therapy of leukemia: 3 decades of development[J]. Blood, 2009, 113(16): 3655-3665. [2] Pierce JN, Stein S. Multiple diversity with onindependent fading[J]. Proc IRE, 1960, 48(1): 89-104. [3] Warrell RP Jr. Retinoid resistance in acute promyelocytic leukemia: new mechanisms, strategies, and implications[J]. Blood, 1993, 82: 1949-1953. [4] De Santis GC, Madeira MIA, de Oliveira LCO, et al. Cardiac stunning as a manifestation of ATRA differentiation syndrome in acute promyelocytic leukemia[J]. Med Oncol, 2012, 29: 248-250. [5] Tohda S, Curtis JE, McCulloch EA, et al. Comparison of the effects of all-trans and cis-retinoic acid on the blast stem cells of acute myeloblastic leukemia in culture[J]. Leukemia, 1992, 6(7): 656-661. [6] Choi BM, Park R, Pae HO, et al. Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells[J]. Pharmacol Toxicol, 2000, 86(2): 53–58. [7] Kassi E, Papoutsi Z, Pratsinis H, et al. Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells[J]. Cancer Res Clin Oncol, 2007, 133: 493–500. [8] Hsu YL, Kuo PL, Lin CC: Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells[J]. Life Sci, 2004, 75(19): 2303–2316. [9] Achiwa Y, Hasegawa K, Komiya T, et al. Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer SNG-II cells[J]. Oncol Rep, 2005, 13(1): 51-57. [10] Manu KA, Kuttan G. Ursolic acid induces apoptosis by activating p53 and caspase-3 gene expressions and suppressing NF-kappa B mediated activation of bcl-2 in B16F-10 melanoma cells[J]. Int Immunopharmacol, 2008, 8(7): 974-981. [11] Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts[J]. Genes Dev, 1999, 13(22): 2905-2927.
[12] Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004, 1697(1-2): 3-16. [13] Matkovic K, Brugnoli F, Bertagnolo V, et al. The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells[J]. Leukemia, 2006, 20: 941-951. [14] Williams MR, Arthur JS, Balendran A, et al. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells[J]. Curr Biol, 2000, 10(8): 439-448. [15] Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[J]. Science, 2005, 307(5712): 1098-1101. [16] Poon KH, Zhang J, Wang C, et al. Betulinic acid enhances 1α, 25-dihydroxyvitamin D3-induced differentiation in human HL-60 promyelocytic leukemia cells[J]. Anticancer Drugs, 2004, 15(6): 619-624. [17] Brackman D, Lund F, Aarskog D. Expression of leukocyte differentiation antigens during the differentiation of HL-60 cells induced by 1, 25-dihydroxyvitamin D3: comparison with the maturation of normal monocytic and granulocytic bone marrow cells[J]. Leukoc Biol, 1995, 58(5): 547-555. [18] Hmama Z, Nandan D, Sly L, et al. 1alpha,25- dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex[J]. Exp Med, 1999, 190(11): 1583-1594. [19] Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1[J]. EMBO, 1996, 15(23): 6541-6551. [20] Yamaguchi H, Noshita T, Kidachi Y, et al. Isolation of ursolic acid from apple peels and its specific efficacy as a potent antitumor agent[J]. Health Sci, 2008, 54: 654-660. [21] Tian Z, Lin G, Zheng RX, et al. Anti-hepatoma activity and mechanism of ursolic acid and its derivatives isolated from Aralia decaisneana[J]. World J Gastroenterol, 2006, 12(6): 874-879. [22] Ishida S, Shigemoto-Mogami Y, Shinozaki Y, et al. Differential modulation of PI3- kinase/Akt pathway during all-trans retinoic acid-and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis[J]. Biochem Pharmacol, 2004, 68(11): 2177- 2186. [23]Kubohara Y, Hosaka K. The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells[J]. BBRC, 1999, 263(3): 790-796. [24] Wang J, Zhao Y, Kauss MA, et al. Akt regulates vitamin D3-induced leukemia cell functional differentiation via Raf/MEK/ERK MAPK signaling[J]. Eur J Cell Biol, 2009, 88(2): 103-115. [25] Missiroli S, Etro D, Buontempo F, et al. Nuclear translocation of active AKT is required for erythroid differentiation in erythropoietin-treated K562 erythroleukemia cells[J]. Int J Biochem Cell Biol, 2009, 41(3): 570-577.
Cite this article as: DENG Lin, ZHANG Rui, TANG Feng, LI Chen, XING Ying-Ying, XI Tao. Ursolic acid induces U937 cells differentiation by PI3K/Akt pathway activation [J]. Chinese Journal of Natural Medicines, 2014, 12(1): 15-19
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