- Email: [email protected]
Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells
Biochemical and Biophysical Research Communications 394 (2010) 476–481
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells Jaleel Kizhakkayil, Faisal Thayyullathil, Shahanas Chathoth, Abdulkader Hago, Mahendra Patel, Sehamuddin Galadari * Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain, United Arab Emirates
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Article history: Received 28 January 2010 Available online 6 February 2010 Keywords: Akt phosphorylation Curcumin MCF-7 cells PI3K inhibitor Apoptosis
a b s t r a c t Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3b (GSK3b), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3b. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells. Ó 2010 Elsevier Inc. All rights reserved.
Introduction Curcumin, or diferuloylmethane, is the major yellow pigment extracted from turmeric (Curcuma longa) and is commonly used as a flavoring agent in food. Curcumin has been reported to possess anti-inflammatory and anti-oxidant activities [1], and it has been reported as a potent inhibitor of mutagenesis [2] and carcinogenesis [3]. Curcumin is also a potent chemo-preventive agent inhibiting tumor promotion against skin, oral, intestine and colorectal cancer [4]. Importantly, curcumin has been shown to induce apoptosis in malignant cancer cell lines including HL60, K562, MCF-7, and HeLa cells [5–7]. The effect of curcumin, such as, inhibition of nitric oxide synthase, receptor tyrosine kinase and protein kinase C [8], Akt [9–11], AP [12], cyclin D1 [13] and activation of p53 [14], has been reported. Akt is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases (Akt 1, 2, 3). Full activation of Akt requires phosphorylation at Ser473 and Thr308 by PDK1. Once activated, Akt in turn regulates a wide range of target proteins such as Bad [15], caspase-9 [16], glycogen synthase kinase (GSK)-3b [17] and XIAP [18], which regulate cell survival, proliferation and growth. It has been suggested that Akt inhibits apoptosis
* Corresponding author. Fax: +971 37672033. E-mail address: [email protected] (S. Galadari). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.01.132
at a pre-mitochondrial step, since it inhibits cytochrome c release, and causes alteration of mitochondrial membrane potential [19]. Previously, we showed that curcumin-induced ROS generation leads to caspase dependant and independent apoptosis in L929 cells [14]. In this study, we report on modulation of curcumin-induced Akt phosphorylation by PI3K inhibitor (LY290042). We found that PI3K inhibitor LY290042 is capable of inhibiting curcumin-induced phosphorylation of Akt, hence, sensitizing curcumininduced apoptosis in MCF-7 cells. Materials and methods Reagents. Curcumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), proteinase K and RNase are purchased from Sigma chemical Co. (St. Louis, MO). LY294002 was purchased from Alexis biochemicals (USA). RPMI 1640 GlutaMAX medium, fetal bovine serum (FBS) and trypsin EDTA were brought from GIBCO BRL (Grand Island, NY). Anti-actin, anti-Bax, anti-Gsk3ab and antip53, antibodies were purchased from Santa Cruz biotechnology, USA. Anti-PARP, anti-Akt, anti-pAkt Ser473and anti-pGsk3b Ser219 was purchased from Cell signaling technology, USA. Cell culture and curcumin treatment. The MCF-7 cells (ATCC) were grown in RPMI 1640 containing GlutaMAX medium supplemented with 10% (V/V) heat inactivated fetal bovine serum without antibiotics in an incubator containing a humidified atmo-
J. Kizhakkayil et al. / Biochemical and Biophysical Research Communications 394 (2010) 476–481
sphere of 95% air and 5% CO2 at 37 °C. Curcumin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 20 mM and was stored in a dark colored bottle at 20 °C. The stock was diluted to the required concentration with DMSO when needed. Prior to curcumin treatment cells were grown to about 80% confluence, and then exposed to curcumin at different concentrations (0–100 lM) and for a different period of time (0–24 h). Cells grown in a medium containing an equivalent amount of DMSO without curcumin served as control. Measurement of cytotoxicity by MTT assay. Cytotoxicity assays were carried out as described previously without any modifica-
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tions [16]. Cells were grown in 96-well microtiter plates (10,000 cells/well) and they were incubated for 24 h with or without different concentrations of curcumin (0–100 lM). At the required time point 100 ll media were removed and 25 ll of MTT (5 mg/ml) was added to each well. The plates were incubated for a further 4 h at 37 °C. After incubation the plates were centrifuged at 1500 rpm for 5 min and the media were removed from all the wells. The formazan crystals that formed were then solubilized in a 200 ll of DMSO. The colored solution was quantified at 570 nm by using Perkin Elmer Victor-3 Spectrophotometer. The cytotoxicity was expressed as percentage over control.
Fig. 1. Effect of curcumin on morphological characteristics and cell viability of MCF-7 cells. (A) MCF-7 cells were treated with either vehicle or the indicated concentrations of curcumin for 24 h, (B) dose dependent effect of curcumin treatment for 24 h on MCF-7 cell viability determined by MTT assay, and (C) time course of the effect of 40 lM curcumin treatment on MCF-7 cell viability using MTT assay. Data shown represent the averages (±SD) of three independent experiments. (D) Dose dependent DNA fragmentation pattern following treatment of MCF-7 cells with curcumin for 24 h. DNA was extracted and analyzed on a 1.2% agarose gel as explained in the ‘‘Materials and methods”.
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DNA fragmentation. Apoptotic DNA fragments were isolated from the apoptotic cells as described previously [9]. MCF-7 cells were grown at a density of 3 106 cells per plate and incubated with various concentrations of curcumin. Cells were then washed with PBS and incubated with 200 ll of lysis buffer (50 mM Tris– HCl (pH 7.5), 3% nonionic detergent IGPAL CA-630 [(octylphenoxy) polyethoxyethanol], and 20 mM EDTA) for 10 min. The samples were centrifuged at 1000g for 5 min in order to collect the supernatant which contain apoptotic DNA fragment. SDS (50 ll, 5%) was added and the supernatants were incubated with 0.4 lg/ ml RNase at 56 °C for 2 h to remove the cellular RNA. Proteinase K (1.5 lg/ml) was then added to the supernatant at 56 °C and it was further incubated for 2 h to remove the proteins. The DNA was then precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volume of ice-cold absolute ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and then air-dried. The dried pellet were resuspended in 20 ll TE buffer (10 mM Tris–HCl, pH 7.5, and 0.1 mM EDTA) and incubated at 65 °C for 5 min. Finally the resuspended DNA was subjected to electrophoresis on a 1.2% agarose gel at a constant voltage of 40 V for 3–4 h. Western blotting. Cells were washed with ice-cold phosphate saline buffer (PBS) and lysed with 200 ll of lysis buffer [50 mM Tris HCl (pH 7.4)), 1% NP-40, 40 mM NaF, 10 mM NaCl, 10 mM Na3VO4, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), and EDTA-free Roshe protease inhibitor tablets per 20 ml
buffer]. The cell lysates were centrifuged at 10,000g for 15 min. Total protein, determined by the Bio-Rad protein assay, was mixed with 6 loading buffer and boiled at 100 °C for 3 min. Samples at 40 lg/lane were resolved by SDS–PAGE and separated proteins were transferred onto nitrocellulose membranes by the wet transfer method using a Bio-Rad electrotransfer apparatus. Following transfer, the blots were blocked with 5% nonfat milk in Tris buffer saline containing 0.1% Tween 20 and then incubated with primary antibodies followed by secondary antibodies. Proteins were visualized by using the enhanced chemiluminescence system. Western blot data presented are representative of those obtained in at least three separate experiments. Result Effect of curcumin on MCF-7 cell viability The effect of different curcumin doses (0–100 lm) on MCF-7 cell morphology was examined. After a 24 h period, curcumin treatment caused MCF-7 cell shrinkage, rounding and partial detachment, thus, demonstrating the cytotoxic effects of curcumin on MCF-7 cells as shown in Fig. 1A. On assaying the effect of curcumin on cell viability by MTT assay, we observed a decrease in cell viability (Fig. 1B). At 40 lM curcumin concentration, significant loss of viability can be detected during the 0–24 h treatment period. It is noteworthy that curcumin-mediated reduction of cell viability was dose and time dependent (Fig. 1B and C). In order to confirm if MCF-7 cells were undergoing apoptosis, cells were treated with various concentrations of curcumin, and DNA was extracted and analyzed for laddering by using agarose gel electrophoresis. A typical DNA ladder pattern of inter-nucleosomal fragmentation, particularly at high curcumin concentrations, was observed (Fig. 1D). Effect of curcumin on PARP cleavage Previous studies have identified that caspase dependant cleavage of PARP is the prime hallmark for caspase dependant apoptosis [16]. Therefore, we examined the effect of curcumin on PARP. As can be seen in Fig. 2A and B, curcumin induced the cleavage of PARP in a dose and time dependent manner. Hence, confirming that curcumin induced reduction in the MCF-7 cells viability is mediated via caspase-dependent apoptosis.
Fig. 2. Curcumin-induced PARP cleavage and induction of p53 and Bax in MCF-7 cells. (A) MCF-7 cells were treated with 0–60 lM curcumin for 24 h time period, and (B) MCF-7 cells were treated with 40 lM curcumin for different time periods. Equal amount of lysates (30 lg) were subjected to electrophoresis and PARP cleavage was analyzed by western blot. The 86 kDa proteolytic PARP cleavage fragment is indicated by arrow. (C) MCF-7 cells were treated with 40 lM of curcumin for indicated time points and the expression levels of p53 and Bax proteins were measured by western blot analysis. Loading accuracy is confirmed by stripping and re-blotting with actin.
Fig. 3. Curcumin-induced Akt phosphorylation in MCF-7 cells. (A) MCF-7 cells were treated with 40 lM of curcumin for the indicated time points and the phosphorylation of Akt protein, and (B) GSK3b were measured by western blot analysis.
J. Kizhakkayil et al. / Biochemical and Biophysical Research Communications 394 (2010) 476–481
Effect of curcumin on p53 and Bax protein induction The protein p53 plays a very important role in cell cycle arrest, and in apoptosis induced by a broad range of agents. In order to examine expression characteristics of p53 in curcumin treated MCF-7 cells we employed western blot analysis. Fig. 2A illustrates that treatment of MCF-7 cells with curcumin induced an increase in p53 levels as early as 3 h, and it reached a maximum by 6 h. In order to further understand the mechanism of curcumin-induced p53 mediated apoptosis in MCF-7 cells, we examined the expression of downstream targets of p53 including Bax and p21. It can be observed in Fig. 2C that Bax initially increases by 3 h following curcumin treatment and maximizing by 12 h. On the other hand, p21 expression level did not change (data not shown). Curcumin up-regulates Akt phosphorylation in MCF-7 cells It is now well established that activation/inactivation of Akt is the key determinant in defining sensitivity to chemotherapeutic drugs. To confirm this we treated MCF-7 cells with 40 lM curcumin for different time periods, and the collected cell lysates were subjected to Western blot analysis for phospho-Akt. The result in
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Fig. 3A showed that curcumin increases the phosphorylation of Akt at Ser473 in a time dependant manner, without effecting total Akt expression. Glycogen synthase kinase 3b, a downstream Akt substrate, mediates apoptosis in response to various stimuli. GSK3b is inhibited by Akt once it is phosphorylated by Akt on serine residue 9. As shown in Fig. 3B curcumin induced the phosphorylation of GSK3b at Ser9. Thus, Akt phosphorylation at Ser473 is correlating with phosphorylation of GSK3b at ser9. PI3K inhibitor reverses curcumin-induced phosphorylation of Akt and enhances MCF-7 cell death Since curcumin enhances Akt phosphorylation in MCF-7 cells (Fig. 3A), we investigated whether the combined treatment with curcumin and LY290042, a selective PI3K inhibitor, would reverse curcumin-induced phosphorylation of Akt. MCF-7 cells were treated with LY290042 and curcumin for 12 h. LY290042 markedly down regulated curcumin-induced Akt phosphorylation at Ser473 (Fig. 4A). Moreover, LY290042 synergistically enhanced the curcumin-induced apoptosis as measured by MTT assay (Fig. 4B), and PARP cleavage (Fig. 4C). In order to examine the effect of the combined treatment with LY290042 and curcumin on Akt downstream
Fig. 4. Effect of LY290042 on curcumin-induced Akt phosphorylation and MCF-7 cell apoptosis. MCF-7 cells were treated with 50 lM concentration of LY290042 for 1 h prior to treatment with curcumin (40 lM) for 12 h. The cell lysate samples were probed with (A) anti-phospho-Akt and Akt, (C) PARP, and (D) phospho-GSK3b and GSK3ab (D) antibodies. (B) MCF-7 cells were treated with 50 lM concentration of LY290042 for 1 h prior to treatment with curcumin (40 lM) for 12 h and MTT assay was performed as described in the ‘‘Materials and methods”. Data shown represent the averages of three independent experiments.
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targets, GSK3b was investigated. As shown in Fig. 4D, the combined treatment with LY290042 and curcumin down regulates curcumininduced GSK3b phosphorylation at Ser9. These results suggest that the combined treatment of MCF-7 cells with LY290042 and curcumin, induces the inactivation of Akt and activation of GSK3b, and subsequently promotes MCF-7 apoptosis.
Acknowledgments This work was financially supported by grants from The Terry Fox Foundation for Cancer Research (UAE-2008), and in part the Sheikh Hamdan Award for Medical Sciences, and grants from the Faculty of Medicine and Health Sciences, United Arab Emirates University.
Discussion References In order to better understand the mechanism of apoptosis induced by curcumin, we investigated the effects of this compound on MCF-7 cells. Curcumin is known to have anti-proliferative, anti-metastatic, anti-inflammatory, and anti-oxidant effects [1,20,21]. In fact, curcumin is under preclinical trial evaluation as a cancer preventive drug [22]. The molecular mechanism underlying curcumin-induced apoptosis is currently being elucidated. Indeed, the role of NF-jB in curcumin-induced apoptosis is well characterized [23]. Other reported signaling effects of curcumin include: inhibition of nitric oxide synthase, receptor tyrosine kinase, protein kinase C [8], Akt [9–11], NF-jB [24], cyclin D1 [13] and activation of p53. Importantly, curcumin is one of the most widely characterized phytochemical, exhibiting anticancer properties against a variety of cancer cells. Curcumin causes morphological changes to MCF-7 cells (Fig. 1A), and it reduces MCF-7 cell viability in a dose and time dependent manner (Fig. 1B and C). This implies the existence of a specific effect of curcumin on MCF-7 cells. The typical DNA laddering pattern of inter-nucleosomal fragmentation, particularly observed at high concentrations of curcumin, confirm that MCF-7 cells are undergoing apoptosis (Fig. 1D). The DNA laddering together with the PARP cleavage data reveal that curcumin induces apoptotic MCF-7 cell death. Tumor cells often evade apoptosis by over expressing antiapoptotic proteins such as Akt, Bcl2, and NF-jB which give them a survival advantage [25–27]. Some conventional chemotherapeutic drugs, such as taxol [23] and doxorubicin [28], cause up-regulation of survival signals, necessitating increase of the effective dose of treatment. Several research groups, including our laboratory, have shown that curcumin promotes apoptosis by interfering with cell survival signaling pathways [7,10,14,29]. Moreover, it has been reported that curcumin inhibits taxol-induced up-regulation of Akt phosphorylation in HeLa cells [30]. Interestingly, in the present study we observed that at apoptosis inducing concentration, curcumin causes phosphorylation of Akt at Ser473. Thus, we investigated the effect of a potent PI3K inhibitor (LY290042) in order to sensitize curcumin-induced apoptosis by reversing Akt phosphorylation. It has been previously reported that treatment with LY290042 resulted in the enhancement of taxol-induced cytotoxicity [31]. Activated Akt protects cells from apoptosis by suppressing NF-jB activity [32], Bax translocation to mitochondria [33], and caspase-9 cleavage. Caspase-9 has been reported to be directly phosphorylated by activated Akt, and its pro-apoptotic activity being suppressed as a result of this phosphorylation [17]. It has been shown that curcumin inhibits camptothecin-mediated apoptosis in MCF-7 cells [34], this may be achieved by the activation of the Akt survival pathway. LY290042 inhibits Akt phosphorylation induced by curcumin, and activates its downstream target GSK3b, which sensitizes the curcumin-induced apoptosis. Taken together, these results demonstrate for the first time that blocking the PI3K/ Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells. Moreover, these findings give rise to the possibility of the negative impact of curcumin on curcumin consuming patient who receive breast cancer chemotherapy. However, further in vivo studies are required to prove this assumption.
[1] A.J. Ruby, G. Kuttan, K.D. Babu, K.N. Rajasekharan, R. Kuttan, Anti-tumour and antioxidant activity of natural curcuminoids, Cancer Lett. 94 (1995) 79–83. [2] K.B. Soni, M. Lahiri, P. Chackradeo, S.V. Bhide, R. Kuttan, Protective effect of food additives on aflatoxin-induced mutagenicity and hepatocarcinogenicity, Cancer Lett. 115 (1997) 129–133. [3] M.T. Huang, Y.R. Lou, J.G. Xie, W.Ma.Y.P. Lu, P. Yen, B.T. Zhu, H. Newmark, C.T. Ho, Effect of dietary curcumin and dibenzoylmethane on formation of 7,12dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/ leukemias in Sencar mice, Carcinogenesis 19 (1998) 1697–1700. [4] M.T. Huang, R.C. Smart, C.Q. Wong, A.H. Conney, Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate, Cancer Res. 48 (1988) 5941– 5946. [5] M.C. Jiang, H.F. Yang-Yen, J.J. Yen, J.K. Lin, Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines, Nutr. Cancer 26 (1996) 111–120. [6] M.L. Kuo, T.S. Huang, J.K. Lin, Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells, Biochim. Biophys. Acta 1317 (1996) 95–100. [7] R.J. Anto, A. Mukhopadhyay, K. Denning, B.B. Aggarwal, Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl, Carcinogenesis 23 (2002) 143–150. [8] J.Y. Liu, S.J. Lin, J.K. Lin, Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells, Carcinogenesis 14 (1993) 857–861. [9] M.H. Pan, S.Y. Lin-Shiau, J.K. Lin, Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkappaB kinase and NFkappaB activation in macrophages, Biochem. Pharmacol. 60 (2000) 1665–1676. [10] S. Singh, B.B. Aggarwal, Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane), J. Biol. Chem. 270 (1995) 24995–25000. [11] S. Aggarwal, H. Ichikawa, Y. Takada, S.K. Sandur, S. Shishodia, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation, Mol. Pharmacol. 69 (2006) 195–206. [12] M. Tomita, H. Kawakami, J.N. Uchihara, T. Okudaira, M. Masuda, N. Takasu, T. Matsuda, T. Ohta, Y. Tanaka, N. Mori, Curcumin suppresses constitutive activation of AP-1 by downregulation of JunD protein in HTLV-1-infected Tcell lines, Leuk. Res. 30 (2006) 313–321. [13] A. Mukhopadhyay, S. Banerjee, L. J Stafford, C. Xia, M. Liu, B.B. Aggarwal, Curcumin-induced suppression of cell proliferation correlates with downregulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation, Oncogene 21 (2002) 8852–8861. [14] F. Thayyullathil, S. Chathoth, A. Hago, M. Patel, S. Galadari, Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspasedependent and -independent apoptosis in L929 cells, Free Radical Biol. Med. 45 (2008) 1403–1412. [15] T.F. Franke, S.I. Yang, T.O. Chan, K. Datta, A. Kazlauskas, D.K. Morrison, D.R. Kaplan, P.N. Tsichlis, The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase, Cell 81 (1995) 727–736. [16] L. del Peso, M. González-García, C. Page, R. Herrera, G. Nuñez, Interleukin-3induced phosphorylation of BAD through the protein kinase Akt, Science 278 (1997) 687–699. [17] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S. Frisch, J.C. Reed, Regulation of cell death protease caspase-9 by phosphorylation, Science 282 (1998) 1318–1321. [18] H.C. Dan, M. Sun, S. Kaneko, R.I. Feldman, S.V. Nicosia, H.G. Wang, B.K. Tsang, J.Q. Cheng, Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP), J. Biol. Chem. 279 (2004) 5405–5412. [19] S.G. Kennedy, E.S. Kandel, T.K. Cross, N. Hay, Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria, Mol. Cell Biol. 19 (1999) 5800–5810. [20] M.A. Azuine, J.J. Kayal, S.V. Bhide, Protective role of aqueous turmeric extract against mutagenicity of direct-acting carcinogens as well as benzo [alpha] pyrene-induced genotoxicity and carcinogenicity, J. Cancer Res. Clin. Oncol. 118 (1992) 447–452. [21] C.V. Rao, A. Rivenson, B. Simi, B.S. Reddy, Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound, Cancer Res. 55 (1995) 259–266.
J. Kizhakkayil et al. / Biochemical and Biophysical Research Communications 394 (2010) 476–481 [22] T. Kawamori, R. Lubet, V.E. Steele, G.J. Kelloff, R.B. Kaskey, C.V. Rao, B.S. Reddy, Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression stages of colon cancer, Cancer Res. 59 (1999) 597–601. [23] R.J. Anto, T.T. Maliekal, D. Karunagaran, L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis, J. Biol. Chem. 275 (2000) 15601–15614. [24] N. Aravindan, R. Madhusoodhanan, S. Ahmad, D. Johnson, T.S. Herman, Curcumin inhibits NFkappaB mediated radioprotection and modulate apoptosis related genes in human neuroblastoma cells, Cancer Biol. Ther. 7 (2008) 569–576. [25] O. Hermine, C. Haioun, E. Lepage, M.F. d’Agay, J. Briere, C. Lavignac, G. Fillet, G. Salles, J.P. Marolleau, J. Diebold, F. Reyas, P. Gaulard, Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin’s lymphoma. Groupe d’Etude des Lymphomes de l’Adulte (GELA), Blood 87 (1996) 265–272. [26] W. Wang, J.L. Abbruzzese, D.B. Evans, L. Larry, K.R. Cleary, P.J. Chiao, The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells, Clin. Cancer Res. 5 (1999) 119–127. [27] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer, Nat. Rev. Cancer. 2 (2002) 489–501. [28] S. Li, Y. Zhou, R. Wang, H. Zhang, Y. Dong, C. Ip, Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-Akt and its downstream substrates, Mol. Cancer Ther. 6 (2007) 1031– 1038.
481
[29] L.R. Chaudhary, K.A. Hruska, Inhibition of cell survival signal protein kinase B/ Akt by curcumin in human prostate cancer cells, J. Cell Biochem. 89 (2003) 1– 5. [30] S.V. Bava, V.T. Puliappadamba, A. Deepti, A. Nair, D. Karunagaran, R.J. Anto, Sensitization of taxol-induced apoptosis by curcumin involves downregulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization, J. Biol. Chem. 280 (2005) 6301– 6308. [31] D.M. Nguyen, G.A. Chen, R. Reddy, W. Tsai, W.D. Schrump, G. Cole Jr., D.S. Schrump, Potentiation of paclitaxel cytotoxicity in lung and esophageal cancer cells by pharmacologic inhibition of the phosphoinositide 3-kinase/protein kinase B (Akt)-mediated signaling pathway, J. Thorac. Cardiovasc. Surg. 127 (2004) 365–375. [32] S. Pianetti, M. Arsura, R. Romieu-Mourez, R.J. Coffey, G.E. Sonenshein, Her-2/ neu overexpression induces NF-kappaB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IkappaB-alpha that can be inhibited by the tumor suppressor PTEN, Oncogene 20 (2001) 1287–1299. [33] F. Tsuruta, N. Masuyama, Y. Gotoh, The phosphatidylinositol 3-kinase (PI3K)Akt pathway suppresses Bax translocation to mitochondria, J. Biol. Chem. 277 (2002) 14040–14047. [34] S. Somasundaram, N.A. Edmund, D.T. Moore, G.W. Small, Y.Y. Shi, R.Z. Orlowski, Dietary curcumin inhibits chemotherapy-induced apoptosis in models of human breast cancer, Cancer Res. 62 (2002) 3868–3875.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells Jaleel Kizhakkayil, Faisal Thayyullathil, Shahanas Chathoth, Abdulkader Hago, Mahendra Patel, Sehamuddin Galadari * Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain, United Arab Emirates
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Article history: Received 28 January 2010 Available online 6 February 2010 Keywords: Akt phosphorylation Curcumin MCF-7 cells PI3K inhibitor Apoptosis
a b s t r a c t Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3b (GSK3b), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3b. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells. Ó 2010 Elsevier Inc. All rights reserved.
Introduction Curcumin, or diferuloylmethane, is the major yellow pigment extracted from turmeric (Curcuma longa) and is commonly used as a flavoring agent in food. Curcumin has been reported to possess anti-inflammatory and anti-oxidant activities [1], and it has been reported as a potent inhibitor of mutagenesis [2] and carcinogenesis [3]. Curcumin is also a potent chemo-preventive agent inhibiting tumor promotion against skin, oral, intestine and colorectal cancer [4]. Importantly, curcumin has been shown to induce apoptosis in malignant cancer cell lines including HL60, K562, MCF-7, and HeLa cells [5–7]. The effect of curcumin, such as, inhibition of nitric oxide synthase, receptor tyrosine kinase and protein kinase C [8], Akt [9–11], AP [12], cyclin D1 [13] and activation of p53 [14], has been reported. Akt is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases (Akt 1, 2, 3). Full activation of Akt requires phosphorylation at Ser473 and Thr308 by PDK1. Once activated, Akt in turn regulates a wide range of target proteins such as Bad [15], caspase-9 [16], glycogen synthase kinase (GSK)-3b [17] and XIAP [18], which regulate cell survival, proliferation and growth. It has been suggested that Akt inhibits apoptosis
* Corresponding author. Fax: +971 37672033. E-mail address: [email protected] (S. Galadari). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.01.132
at a pre-mitochondrial step, since it inhibits cytochrome c release, and causes alteration of mitochondrial membrane potential [19]. Previously, we showed that curcumin-induced ROS generation leads to caspase dependant and independent apoptosis in L929 cells [14]. In this study, we report on modulation of curcumin-induced Akt phosphorylation by PI3K inhibitor (LY290042). We found that PI3K inhibitor LY290042 is capable of inhibiting curcumin-induced phosphorylation of Akt, hence, sensitizing curcumininduced apoptosis in MCF-7 cells. Materials and methods Reagents. Curcumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), proteinase K and RNase are purchased from Sigma chemical Co. (St. Louis, MO). LY294002 was purchased from Alexis biochemicals (USA). RPMI 1640 GlutaMAX medium, fetal bovine serum (FBS) and trypsin EDTA were brought from GIBCO BRL (Grand Island, NY). Anti-actin, anti-Bax, anti-Gsk3ab and antip53, antibodies were purchased from Santa Cruz biotechnology, USA. Anti-PARP, anti-Akt, anti-pAkt Ser473and anti-pGsk3b Ser219 was purchased from Cell signaling technology, USA. Cell culture and curcumin treatment. The MCF-7 cells (ATCC) were grown in RPMI 1640 containing GlutaMAX medium supplemented with 10% (V/V) heat inactivated fetal bovine serum without antibiotics in an incubator containing a humidified atmo-
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sphere of 95% air and 5% CO2 at 37 °C. Curcumin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 20 mM and was stored in a dark colored bottle at 20 °C. The stock was diluted to the required concentration with DMSO when needed. Prior to curcumin treatment cells were grown to about 80% confluence, and then exposed to curcumin at different concentrations (0–100 lM) and for a different period of time (0–24 h). Cells grown in a medium containing an equivalent amount of DMSO without curcumin served as control. Measurement of cytotoxicity by MTT assay. Cytotoxicity assays were carried out as described previously without any modifica-
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tions [16]. Cells were grown in 96-well microtiter plates (10,000 cells/well) and they were incubated for 24 h with or without different concentrations of curcumin (0–100 lM). At the required time point 100 ll media were removed and 25 ll of MTT (5 mg/ml) was added to each well. The plates were incubated for a further 4 h at 37 °C. After incubation the plates were centrifuged at 1500 rpm for 5 min and the media were removed from all the wells. The formazan crystals that formed were then solubilized in a 200 ll of DMSO. The colored solution was quantified at 570 nm by using Perkin Elmer Victor-3 Spectrophotometer. The cytotoxicity was expressed as percentage over control.
Fig. 1. Effect of curcumin on morphological characteristics and cell viability of MCF-7 cells. (A) MCF-7 cells were treated with either vehicle or the indicated concentrations of curcumin for 24 h, (B) dose dependent effect of curcumin treatment for 24 h on MCF-7 cell viability determined by MTT assay, and (C) time course of the effect of 40 lM curcumin treatment on MCF-7 cell viability using MTT assay. Data shown represent the averages (±SD) of three independent experiments. (D) Dose dependent DNA fragmentation pattern following treatment of MCF-7 cells with curcumin for 24 h. DNA was extracted and analyzed on a 1.2% agarose gel as explained in the ‘‘Materials and methods”.
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DNA fragmentation. Apoptotic DNA fragments were isolated from the apoptotic cells as described previously [9]. MCF-7 cells were grown at a density of 3 106 cells per plate and incubated with various concentrations of curcumin. Cells were then washed with PBS and incubated with 200 ll of lysis buffer (50 mM Tris– HCl (pH 7.5), 3% nonionic detergent IGPAL CA-630 [(octylphenoxy) polyethoxyethanol], and 20 mM EDTA) for 10 min. The samples were centrifuged at 1000g for 5 min in order to collect the supernatant which contain apoptotic DNA fragment. SDS (50 ll, 5%) was added and the supernatants were incubated with 0.4 lg/ ml RNase at 56 °C for 2 h to remove the cellular RNA. Proteinase K (1.5 lg/ml) was then added to the supernatant at 56 °C and it was further incubated for 2 h to remove the proteins. The DNA was then precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volume of ice-cold absolute ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and then air-dried. The dried pellet were resuspended in 20 ll TE buffer (10 mM Tris–HCl, pH 7.5, and 0.1 mM EDTA) and incubated at 65 °C for 5 min. Finally the resuspended DNA was subjected to electrophoresis on a 1.2% agarose gel at a constant voltage of 40 V for 3–4 h. Western blotting. Cells were washed with ice-cold phosphate saline buffer (PBS) and lysed with 200 ll of lysis buffer [50 mM Tris HCl (pH 7.4)), 1% NP-40, 40 mM NaF, 10 mM NaCl, 10 mM Na3VO4, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), and EDTA-free Roshe protease inhibitor tablets per 20 ml
buffer]. The cell lysates were centrifuged at 10,000g for 15 min. Total protein, determined by the Bio-Rad protein assay, was mixed with 6 loading buffer and boiled at 100 °C for 3 min. Samples at 40 lg/lane were resolved by SDS–PAGE and separated proteins were transferred onto nitrocellulose membranes by the wet transfer method using a Bio-Rad electrotransfer apparatus. Following transfer, the blots were blocked with 5% nonfat milk in Tris buffer saline containing 0.1% Tween 20 and then incubated with primary antibodies followed by secondary antibodies. Proteins were visualized by using the enhanced chemiluminescence system. Western blot data presented are representative of those obtained in at least three separate experiments. Result Effect of curcumin on MCF-7 cell viability The effect of different curcumin doses (0–100 lm) on MCF-7 cell morphology was examined. After a 24 h period, curcumin treatment caused MCF-7 cell shrinkage, rounding and partial detachment, thus, demonstrating the cytotoxic effects of curcumin on MCF-7 cells as shown in Fig. 1A. On assaying the effect of curcumin on cell viability by MTT assay, we observed a decrease in cell viability (Fig. 1B). At 40 lM curcumin concentration, significant loss of viability can be detected during the 0–24 h treatment period. It is noteworthy that curcumin-mediated reduction of cell viability was dose and time dependent (Fig. 1B and C). In order to confirm if MCF-7 cells were undergoing apoptosis, cells were treated with various concentrations of curcumin, and DNA was extracted and analyzed for laddering by using agarose gel electrophoresis. A typical DNA ladder pattern of inter-nucleosomal fragmentation, particularly at high curcumin concentrations, was observed (Fig. 1D). Effect of curcumin on PARP cleavage Previous studies have identified that caspase dependant cleavage of PARP is the prime hallmark for caspase dependant apoptosis [16]. Therefore, we examined the effect of curcumin on PARP. As can be seen in Fig. 2A and B, curcumin induced the cleavage of PARP in a dose and time dependent manner. Hence, confirming that curcumin induced reduction in the MCF-7 cells viability is mediated via caspase-dependent apoptosis.
Fig. 2. Curcumin-induced PARP cleavage and induction of p53 and Bax in MCF-7 cells. (A) MCF-7 cells were treated with 0–60 lM curcumin for 24 h time period, and (B) MCF-7 cells were treated with 40 lM curcumin for different time periods. Equal amount of lysates (30 lg) were subjected to electrophoresis and PARP cleavage was analyzed by western blot. The 86 kDa proteolytic PARP cleavage fragment is indicated by arrow. (C) MCF-7 cells were treated with 40 lM of curcumin for indicated time points and the expression levels of p53 and Bax proteins were measured by western blot analysis. Loading accuracy is confirmed by stripping and re-blotting with actin.
Fig. 3. Curcumin-induced Akt phosphorylation in MCF-7 cells. (A) MCF-7 cells were treated with 40 lM of curcumin for the indicated time points and the phosphorylation of Akt protein, and (B) GSK3b were measured by western blot analysis.
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Effect of curcumin on p53 and Bax protein induction The protein p53 plays a very important role in cell cycle arrest, and in apoptosis induced by a broad range of agents. In order to examine expression characteristics of p53 in curcumin treated MCF-7 cells we employed western blot analysis. Fig. 2A illustrates that treatment of MCF-7 cells with curcumin induced an increase in p53 levels as early as 3 h, and it reached a maximum by 6 h. In order to further understand the mechanism of curcumin-induced p53 mediated apoptosis in MCF-7 cells, we examined the expression of downstream targets of p53 including Bax and p21. It can be observed in Fig. 2C that Bax initially increases by 3 h following curcumin treatment and maximizing by 12 h. On the other hand, p21 expression level did not change (data not shown). Curcumin up-regulates Akt phosphorylation in MCF-7 cells It is now well established that activation/inactivation of Akt is the key determinant in defining sensitivity to chemotherapeutic drugs. To confirm this we treated MCF-7 cells with 40 lM curcumin for different time periods, and the collected cell lysates were subjected to Western blot analysis for phospho-Akt. The result in
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Fig. 3A showed that curcumin increases the phosphorylation of Akt at Ser473 in a time dependant manner, without effecting total Akt expression. Glycogen synthase kinase 3b, a downstream Akt substrate, mediates apoptosis in response to various stimuli. GSK3b is inhibited by Akt once it is phosphorylated by Akt on serine residue 9. As shown in Fig. 3B curcumin induced the phosphorylation of GSK3b at Ser9. Thus, Akt phosphorylation at Ser473 is correlating with phosphorylation of GSK3b at ser9. PI3K inhibitor reverses curcumin-induced phosphorylation of Akt and enhances MCF-7 cell death Since curcumin enhances Akt phosphorylation in MCF-7 cells (Fig. 3A), we investigated whether the combined treatment with curcumin and LY290042, a selective PI3K inhibitor, would reverse curcumin-induced phosphorylation of Akt. MCF-7 cells were treated with LY290042 and curcumin for 12 h. LY290042 markedly down regulated curcumin-induced Akt phosphorylation at Ser473 (Fig. 4A). Moreover, LY290042 synergistically enhanced the curcumin-induced apoptosis as measured by MTT assay (Fig. 4B), and PARP cleavage (Fig. 4C). In order to examine the effect of the combined treatment with LY290042 and curcumin on Akt downstream
Fig. 4. Effect of LY290042 on curcumin-induced Akt phosphorylation and MCF-7 cell apoptosis. MCF-7 cells were treated with 50 lM concentration of LY290042 for 1 h prior to treatment with curcumin (40 lM) for 12 h. The cell lysate samples were probed with (A) anti-phospho-Akt and Akt, (C) PARP, and (D) phospho-GSK3b and GSK3ab (D) antibodies. (B) MCF-7 cells were treated with 50 lM concentration of LY290042 for 1 h prior to treatment with curcumin (40 lM) for 12 h and MTT assay was performed as described in the ‘‘Materials and methods”. Data shown represent the averages of three independent experiments.
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targets, GSK3b was investigated. As shown in Fig. 4D, the combined treatment with LY290042 and curcumin down regulates curcumininduced GSK3b phosphorylation at Ser9. These results suggest that the combined treatment of MCF-7 cells with LY290042 and curcumin, induces the inactivation of Akt and activation of GSK3b, and subsequently promotes MCF-7 apoptosis.
Acknowledgments This work was financially supported by grants from The Terry Fox Foundation for Cancer Research (UAE-2008), and in part the Sheikh Hamdan Award for Medical Sciences, and grants from the Faculty of Medicine and Health Sciences, United Arab Emirates University.
Discussion References In order to better understand the mechanism of apoptosis induced by curcumin, we investigated the effects of this compound on MCF-7 cells. Curcumin is known to have anti-proliferative, anti-metastatic, anti-inflammatory, and anti-oxidant effects [1,20,21]. In fact, curcumin is under preclinical trial evaluation as a cancer preventive drug [22]. The molecular mechanism underlying curcumin-induced apoptosis is currently being elucidated. Indeed, the role of NF-jB in curcumin-induced apoptosis is well characterized [23]. Other reported signaling effects of curcumin include: inhibition of nitric oxide synthase, receptor tyrosine kinase, protein kinase C [8], Akt [9–11], NF-jB [24], cyclin D1 [13] and activation of p53. Importantly, curcumin is one of the most widely characterized phytochemical, exhibiting anticancer properties against a variety of cancer cells. Curcumin causes morphological changes to MCF-7 cells (Fig. 1A), and it reduces MCF-7 cell viability in a dose and time dependent manner (Fig. 1B and C). This implies the existence of a specific effect of curcumin on MCF-7 cells. The typical DNA laddering pattern of inter-nucleosomal fragmentation, particularly observed at high concentrations of curcumin, confirm that MCF-7 cells are undergoing apoptosis (Fig. 1D). The DNA laddering together with the PARP cleavage data reveal that curcumin induces apoptotic MCF-7 cell death. Tumor cells often evade apoptosis by over expressing antiapoptotic proteins such as Akt, Bcl2, and NF-jB which give them a survival advantage [25–27]. Some conventional chemotherapeutic drugs, such as taxol [23] and doxorubicin [28], cause up-regulation of survival signals, necessitating increase of the effective dose of treatment. Several research groups, including our laboratory, have shown that curcumin promotes apoptosis by interfering with cell survival signaling pathways [7,10,14,29]. Moreover, it has been reported that curcumin inhibits taxol-induced up-regulation of Akt phosphorylation in HeLa cells [30]. Interestingly, in the present study we observed that at apoptosis inducing concentration, curcumin causes phosphorylation of Akt at Ser473. Thus, we investigated the effect of a potent PI3K inhibitor (LY290042) in order to sensitize curcumin-induced apoptosis by reversing Akt phosphorylation. It has been previously reported that treatment with LY290042 resulted in the enhancement of taxol-induced cytotoxicity [31]. Activated Akt protects cells from apoptosis by suppressing NF-jB activity [32], Bax translocation to mitochondria [33], and caspase-9 cleavage. Caspase-9 has been reported to be directly phosphorylated by activated Akt, and its pro-apoptotic activity being suppressed as a result of this phosphorylation [17]. It has been shown that curcumin inhibits camptothecin-mediated apoptosis in MCF-7 cells [34], this may be achieved by the activation of the Akt survival pathway. LY290042 inhibits Akt phosphorylation induced by curcumin, and activates its downstream target GSK3b, which sensitizes the curcumin-induced apoptosis. Taken together, these results demonstrate for the first time that blocking the PI3K/ Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells. Moreover, these findings give rise to the possibility of the negative impact of curcumin on curcumin consuming patient who receive breast cancer chemotherapy. However, further in vivo studies are required to prove this assumption.
[1] A.J. Ruby, G. Kuttan, K.D. Babu, K.N. Rajasekharan, R. Kuttan, Anti-tumour and antioxidant activity of natural curcuminoids, Cancer Lett. 94 (1995) 79–83. [2] K.B. Soni, M. Lahiri, P. Chackradeo, S.V. Bhide, R. Kuttan, Protective effect of food additives on aflatoxin-induced mutagenicity and hepatocarcinogenicity, Cancer Lett. 115 (1997) 129–133. [3] M.T. Huang, Y.R. Lou, J.G. Xie, W.Ma.Y.P. Lu, P. Yen, B.T. Zhu, H. Newmark, C.T. Ho, Effect of dietary curcumin and dibenzoylmethane on formation of 7,12dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/ leukemias in Sencar mice, Carcinogenesis 19 (1998) 1697–1700. [4] M.T. Huang, R.C. Smart, C.Q. Wong, A.H. Conney, Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate, Cancer Res. 48 (1988) 5941– 5946. [5] M.C. Jiang, H.F. Yang-Yen, J.J. Yen, J.K. Lin, Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines, Nutr. Cancer 26 (1996) 111–120. [6] M.L. Kuo, T.S. Huang, J.K. Lin, Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells, Biochim. Biophys. Acta 1317 (1996) 95–100. [7] R.J. Anto, A. Mukhopadhyay, K. Denning, B.B. Aggarwal, Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl, Carcinogenesis 23 (2002) 143–150. [8] J.Y. Liu, S.J. Lin, J.K. Lin, Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells, Carcinogenesis 14 (1993) 857–861. [9] M.H. Pan, S.Y. Lin-Shiau, J.K. Lin, Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkappaB kinase and NFkappaB activation in macrophages, Biochem. Pharmacol. 60 (2000) 1665–1676. [10] S. Singh, B.B. Aggarwal, Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane), J. Biol. Chem. 270 (1995) 24995–25000. [11] S. Aggarwal, H. Ichikawa, Y. Takada, S.K. Sandur, S. Shishodia, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation, Mol. Pharmacol. 69 (2006) 195–206. [12] M. Tomita, H. Kawakami, J.N. Uchihara, T. Okudaira, M. Masuda, N. Takasu, T. Matsuda, T. Ohta, Y. Tanaka, N. Mori, Curcumin suppresses constitutive activation of AP-1 by downregulation of JunD protein in HTLV-1-infected Tcell lines, Leuk. Res. 30 (2006) 313–321. [13] A. Mukhopadhyay, S. Banerjee, L. J Stafford, C. Xia, M. Liu, B.B. Aggarwal, Curcumin-induced suppression of cell proliferation correlates with downregulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation, Oncogene 21 (2002) 8852–8861. [14] F. Thayyullathil, S. Chathoth, A. Hago, M. Patel, S. Galadari, Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspasedependent and -independent apoptosis in L929 cells, Free Radical Biol. Med. 45 (2008) 1403–1412. [15] T.F. Franke, S.I. Yang, T.O. Chan, K. Datta, A. Kazlauskas, D.K. Morrison, D.R. Kaplan, P.N. Tsichlis, The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase, Cell 81 (1995) 727–736. [16] L. del Peso, M. González-García, C. Page, R. Herrera, G. Nuñez, Interleukin-3induced phosphorylation of BAD through the protein kinase Akt, Science 278 (1997) 687–699. [17] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S. Frisch, J.C. Reed, Regulation of cell death protease caspase-9 by phosphorylation, Science 282 (1998) 1318–1321. [18] H.C. Dan, M. Sun, S. Kaneko, R.I. Feldman, S.V. Nicosia, H.G. Wang, B.K. Tsang, J.Q. Cheng, Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP), J. Biol. Chem. 279 (2004) 5405–5412. [19] S.G. Kennedy, E.S. Kandel, T.K. Cross, N. Hay, Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria, Mol. Cell Biol. 19 (1999) 5800–5810. [20] M.A. Azuine, J.J. Kayal, S.V. Bhide, Protective role of aqueous turmeric extract against mutagenicity of direct-acting carcinogens as well as benzo [alpha] pyrene-induced genotoxicity and carcinogenicity, J. Cancer Res. Clin. Oncol. 118 (1992) 447–452. [21] C.V. Rao, A. Rivenson, B. Simi, B.S. Reddy, Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound, Cancer Res. 55 (1995) 259–266.
J. Kizhakkayil et al. / Biochemical and Biophysical Research Communications 394 (2010) 476–481 [22] T. Kawamori, R. Lubet, V.E. Steele, G.J. Kelloff, R.B. Kaskey, C.V. Rao, B.S. Reddy, Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression stages of colon cancer, Cancer Res. 59 (1999) 597–601. [23] R.J. Anto, T.T. Maliekal, D. Karunagaran, L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis, J. Biol. Chem. 275 (2000) 15601–15614. [24] N. Aravindan, R. Madhusoodhanan, S. Ahmad, D. Johnson, T.S. Herman, Curcumin inhibits NFkappaB mediated radioprotection and modulate apoptosis related genes in human neuroblastoma cells, Cancer Biol. Ther. 7 (2008) 569–576. [25] O. Hermine, C. Haioun, E. Lepage, M.F. d’Agay, J. Briere, C. Lavignac, G. Fillet, G. Salles, J.P. Marolleau, J. Diebold, F. Reyas, P. Gaulard, Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin’s lymphoma. Groupe d’Etude des Lymphomes de l’Adulte (GELA), Blood 87 (1996) 265–272. [26] W. Wang, J.L. Abbruzzese, D.B. Evans, L. Larry, K.R. Cleary, P.J. Chiao, The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells, Clin. Cancer Res. 5 (1999) 119–127. [27] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer, Nat. Rev. Cancer. 2 (2002) 489–501. [28] S. Li, Y. Zhou, R. Wang, H. Zhang, Y. Dong, C. Ip, Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-Akt and its downstream substrates, Mol. Cancer Ther. 6 (2007) 1031– 1038.
481
[29] L.R. Chaudhary, K.A. Hruska, Inhibition of cell survival signal protein kinase B/ Akt by curcumin in human prostate cancer cells, J. Cell Biochem. 89 (2003) 1– 5. [30] S.V. Bava, V.T. Puliappadamba, A. Deepti, A. Nair, D. Karunagaran, R.J. Anto, Sensitization of taxol-induced apoptosis by curcumin involves downregulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization, J. Biol. Chem. 280 (2005) 6301– 6308. [31] D.M. Nguyen, G.A. Chen, R. Reddy, W. Tsai, W.D. Schrump, G. Cole Jr., D.S. Schrump, Potentiation of paclitaxel cytotoxicity in lung and esophageal cancer cells by pharmacologic inhibition of the phosphoinositide 3-kinase/protein kinase B (Akt)-mediated signaling pathway, J. Thorac. Cardiovasc. Surg. 127 (2004) 365–375. [32] S. Pianetti, M. Arsura, R. Romieu-Mourez, R.J. Coffey, G.E. Sonenshein, Her-2/ neu overexpression induces NF-kappaB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IkappaB-alpha that can be inhibited by the tumor suppressor PTEN, Oncogene 20 (2001) 1287–1299. [33] F. Tsuruta, N. Masuyama, Y. Gotoh, The phosphatidylinositol 3-kinase (PI3K)Akt pathway suppresses Bax translocation to mitochondria, J. Biol. Chem. 277 (2002) 14040–14047. [34] S. Somasundaram, N.A. Edmund, D.T. Moore, G.W. Small, Y.Y. Shi, R.Z. Orlowski, Dietary curcumin inhibits chemotherapy-induced apoptosis in models of human breast cancer, Cancer Res. 62 (2002) 3868–3875.