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Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway
European Journal of Pharmacology 668 (2011) 127–132
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway Abdelazim Ibrahim a, Abdelmoniem El-meligy a, Gina Lungu b, Hamdy Fetaih a, Amina Dessouki a, George Stoica b, Rola Barhoumi c,⁎ a b c
Department of Pathology, College of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843–4467, United States Department of Veterinary Integrative BioSciences, Texas A&M University, College Station, TX 77843–4458, United States
a r t i c l e
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Article history: Received 23 February 2011 Received in revised form 10 June 2011 Accepted 23 June 2011 Available online 8 July 2011 Keywords: Curcumin Apoptosis Cancer Mitochondrial calcium Reactive oxygen species production Mitochondrial uniporter inhibitor
a b s t r a c t Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca 2+ homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca2+ accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca 2+ channel blocker, suppressed the curcumin effect on mitochondrial Ca2+. In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca 2+ is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Interest in the use of medicinal plants to treat human disease has been growing rapidly in the last decade, as they are available, cheap, and thought to be safe. Curcumin, a yellow colored polyphenol, is an active principle of the perennial herb Curcuma longa (commonly known as turmeric). In addition to its extensive use as a food additive, turmeric has been used in traditional medicine for treatment of various inflammatory conditions such as arthritis, colitis and hepatitis. Curcumin has also been shown to suppress multiple signaling pathways and inhibit cell proliferation, invasion, metastasis, and angiogenesis (Bengmark et al., 2009; Kunnumakkara et al., 2008). Apoptosis, the process of programmed cell death, is the desired outcome in the testing of potential anticancer drugs. Many studies have proven that curcumin induces cellular apoptosis (Bae et al., 2003; Ramachandran and You, 1999; Su et al., 2006; Thayyullathil et al., 2008; Wang et al., 2009a, 2009b) through multiple signaling pathways, including cell proliferation pathway, cell survival pathway, caspase activation pathway, tumor suppressor pathway, death
⁎ Corresponding author. Tel.: + 1 979 458 1149; fax: +1 979 847 8981. E-mail address: [email protected] (R. Barhoumi). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.06.048
receptor pathway, mitochondrial pathways, and protein kinase pathway (Ravindran et al., 2009). Mitochondria and intracellular calcium play an important role in apoptosis. In response to the apoptotic stimulus, there is an early increase in cytosolic Ca2+ followed by a delayed increase in mitochondrial Ca2+, which in turn induces the opening of permeability transition pores and disrupts mitochondrial membrane potential (MMP). The collapse of MMP along with release of cytochrome c from mitochondria is followed by activation of caspases, nuclear fragmentation and cell death by apoptosis (Caroppi et al., 2009; Smaili et al., 2003). Cytoplasmic calcium chelators (BAPTA-AM) and inhibitors of mitochondrial calcium uptake (ruthenium red) prevent apoptosis (Kruman and Mattson, 1999). In addition, deregulation of Bcl-2 family members, a frequent feature in human malignant diseases and in some cases the reason behind therapy resistance, can block elevation of mitochondrial calcium and inhibit membrane depolarization in cells exposed to apoptotic stimulus (Frenzel et al., 2009). Reactive oxygen species have also been shown to play an equally important role in the mechanism of apoptosis. Cytochrome c release from mitochondria that triggers caspase activation appears to be largely mediated by reactive oxygen species action (Simon et al., 2000). Specifically, in colon cancer cells curcumin induces cytotoxicity and apoptosis which is mediated by the production of reactive oxygen
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species, an increased Ca 2+ in mitochondria, alteration of mitochondrial membrane potential, and induction of caspase-3 activity. Moreover, curcumin promotes the expression of Bax, cytochrome c, p53 and p21, and inhibits the expression of Bcl-2 (Su et al., 2006). We previously evaluated the effects of curcumin and curcumin formulated with phosphatidylcholine (Meriva) in athymic nude mice inoculated with mammary gland adenocarcinoma (ENU 1564) cells (Ibrahim et al., 2010). In this paper, we report the effects of curcumin on the same cells in vitro to identify the mechanisms by which curcumin induces apoptosis in these cells, the role of MMP and Ca 2+, and how they interact to regulate this process. 2. Materials and methods 2.1. Chemicals and cell line Curcumin, monoclonal mouse IgG antibody against beta-actin, Janus green, and Carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemical Co. (St. Louis, Mo). Curcumin was prepared as a stock solution of 40 mM and stored at −20 ° C until use. The ENU1564 tumor cell line used in this study was developed in our laboratory and originated from an N-ethyl-N nitosourea-induced mammary adenocarcinoma in a female Berlin-Druckrey IV (BD-IV) rat. This highly metastatic cell line (Hall and Stoica, 1994) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) (Invitrogen, Carlsbad, CA, USA). The cells were grown at 37 ° C in a humidified incubator containing 5% CO2 in air. Cells were passaged biweekly and used for experiments when in the exponential growth phase. Live/Dead assay kit, rhod-2 AM, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydro-fluorescein diacetate acetyl ester (CMH2DCFDA), YO-PRO and Hoechst were all purchased from Invitrogen. Rabbit polyclonal IgG anti-caspase-3 and mouse monoclonal IgG anti-Bcl-2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). RU-360 and SKF-96365 were purchased from Calbiochem (LA Jolla, CA). 2.2. Uptake of curcumin and viability assessment in ENU1564 cells ENU1564 cells were plated in a 6-well plate for 1 day followed by treatment with different concentrations of curcumin (0–40 μM) for 24 h. Cells were then washed twice with serum free and phenol red free medium. Fluorescence intensity of curcumin was measured using a Biotek plate reader (Biotek Instruments Inc., Winooski, VT, USA) with an excitation and emission wavelength of 457 nm and 510 nm respectively. Cell count per well was then assessed using the Janus green assay to determine the curcumin fluorescence intensity per cell. The effect of curcumin on cell viability was then assessed using the Live/Dead assay, which distinguishes live cells by the presence of ubiquitous intracellular esterase activity determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells and its uniform green fluorescence can be measured with an excitation wavelength of 488 nm and an emission wavelength of 515 nm. The kit also measures dead cells using ethidium homodimer-1 (EthD-1). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids. It can be measured with an excitation wavelength of 488 nm and an emission wavelength of 635 nm. To perform the live/dead assay, cells were loaded with 10 μM calcein-AM and 5 μM EthD-1 for 30 min and then washed. Selected areas from each plate were scanned and images were collected and saved on a BioRad radiance 2000MP laser scanning microscope (Bio-Rad, UK). To quantitate apoptosis in ENU1564 cells following curcumin treatments, cells were loaded with Hoechst 33342 and YO-PRO. YO-
PRO dye enters apoptotic cells and shows green fluorescence. Bluefluorescent Hoechst 33342 brightly stains the condensed chromatin of apoptotic cells and more dimly stains the normal chromatin of living cells. Cells were stained with these dyes according to Invitrogen protocol. Fluorescence emission at 460 nm and 530 nm were measured using excitation of 400 nm for Hoechst and 488 nm for YO-PRO. 2.3. Western blot analysis of pro-caspase3 and Bcl-2 The cells were washed twice with cold Hank's solution and lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and supplemented with a mixture of protease inhibitors (Sigma, St. Louis, MO, USA). The lysates were cleared by centrifugation at 13,000 g at 4 °C for 30 min and the supernatant kept frozen at −80 °C. The protein content of the lysates was determined using Bradford Assay (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard. Proteins (15–30 μg) were separated by 9–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Membranes were incubated for 1 h in blocking buffer (20 mM Tris-HCl-buffered saline containing 5% nonfat milk powder and 0.1% Tween-20) at room temperature, then probed with appropriate antibodies in blocking buffer or blocking buffer including 5% bovine serum albumin instead of 5% nonfat milk overnight at 4 °C. Blots were incubated at 4 °C overnight with anti-BCL-2 (1:200), antipro-caspase-3 (1:200). The blots were washed extensively and then incubated for 1 h with a 1:5,000 dilution of anti-primary antibody purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). After additional washes, the blots were incubated with chemiluminescent substrate, according to the kit instructions (Super Signal West Pico, Pierce, Rockford, IL). 2.4. Effects of curcumin on store operated channels and mitochondrial Ca 2+ in ENU1564 cells The fluorescent probe rhod-2 AM was used to determine the effects of curcumin on mitochondrial calcium. Briefly, ENU1564 cells were seeded into 96 well plates in 8 replicates per treatment. The cells were incubated for 24 h at 37 °C and 5% CO2. Different concentrations of curcumin (0, 25, 30, 35, 40 μM) were added to the cells. Cells were then incubated for 6, 12, and 24 h. Following treatments, cells were washed twice with serum- and phenol red- free medium and labeled with 3 μM rhod-2 AM for 1 h at 37 °C. Cells were then washed twice, and the mitochondrial Ca 2+ was measured using excitation and emission wavelengths of 540 and 590 nm respectively on the Biotek Synergy 4 plate reader. To identify the role of the mitochondrial uniporter in curcumin induced toxicity, cells were incubated with RU-360 (10 μM), an inhibitor of the mitochondrial uniporter, for 30 min prior to curcumin treatment (Nutt et al., 2002). Cells were then washed and treated with different concentrations of curcumin for 24 h. For quantification of rhod-2 fluorescence, each concentration of curcumin was compared to its own control and all rhod-2 fluorescence intensity values were corrected to the cell number per well determined using the Janus green assay as described below. To investigate the role of store operated Ca 2+ channels and extracellular Ca 2+ influx induced by curcumin on the mitochondrial Ca 2+, ENU1564 cells were co-incubated with 10 μM SKF-96365 and curcumin (0–40 μM), prior to mitochondrial Ca 2+ measurements. 2.5. Analysis of mitochondrial membrane potential (MMP) The effects of curcumin on the mitochondrial membrane potential were measured using JC-1. JC-1 is a cationic dye that exhibits potentialdependent accumulation in mitochondria and is characterized by an excitation wavelength of 490 nm and two emission peaks at 539 nm and 597 nm corresponding to monomer (green) and aggregate (red)
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forms of the dye respectively. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (Di Lisa et al., 1995). In a 96 well plate, tumor cells were treated with different concentrations of curcumin (0, 25, 30, 35, 40 μM). Following 24 h of treatment, cells were washed and loaded with 5 μg/ml of JC-1 for 30 min. To determine how much depolarization is occurring in ENU1564 cells due to curcumin treatment, results with JC-1 were compared to a calibration curve obtained with different concentrations of the uncoupling agent CCCP for different time intervals (Zamzami et al., 1995). In addition, the curcumin effects on MMP were analyzed in ENU1564 cells treated with 10 μM of the mitochondrial uniporter inhibitor RU-360 for 30 min to 1 h prior to curcumin treatment. 2.6. Assessment of reactive oxygen species generation Generation of reactive oxygen species in ENU1564 cells, following treatment with different concentrations of curcumin, was assessed using the fluorescent probe CMH2DCFDA (Hempel et al., 1999). Curcumin treated cells were incubated with 5 μM CMH2DCFDA for 30 min at 37 °C. Cells were then washed, excited at 488 nm wavelength and fluorescence emission per well was measured at 515 nm wavelength using a Biotek Synergy 4 Plate Reader. Reactive oxygen species per cell were computed by dividing the fluorescence intensity measured by the number of cells per well determined using the Janus green assay (described below). 2.7. Cell counts For cell counting in wells, cultures were washed twice with PBS and fixed with methanol for 30 min at room temperature. Methanol was completely removed and 1 mg/ml janus green in PBS was added to the cultures for 3 min. Following removal of Janus green, cultures were washed twice with PBS and 100 μl of 50% methanol was added to each well. Cell counts were then determined with a Biotek synergy 4 plate reader using an absorbance of 654 nm (Raspotnig et al., 1999). 2.8. Statistical analysis Quantification of pro-caspase 3 and Bcl-2 bands was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http:// rsb.info.nih.gov/nih-image). Data were presented as mean ± S.D., and statistical comparisons were made using GraphPad Prism version 4.00 for Windows, (GraphPad Software, San Diego, California, USA, www. graphpad.com). A P-value less than 0.05 was considered statistically significant. Mitochondrial Ca 2+ was presented as mean fluorescence intensity ± S.E.M and mitochondrial membrane potential was presented as ratio (red/green) ± S.E.M. For comparison of response with different concentrations of curcumin, one way ANOVA was used followed by Tukey's multiple comparison test. For experiments involving RU-360, two-way ANOVA was used followed by Bonferroni test. Differences between treatments were considered significant at P b 0.05. 3. Results 3.1. Curcumin decreased the viability of ENU1564 cells The uptake of different concentrations of curcumin by ENU1564 cells following 24 h treatments was recorded, as shown in Fig. 1. Using the live/dead assay to evaluate the toxicity of curcumin, the results showed that the number of dead cells, as indicated by the EthD-1 red fluorescence, increases with increasing curcumin concentrations (0–40 μM). Fig. 2 shows the live (green fluorescence) and dead (red fluorescence) cells in a control culture (left panel) and in cultures
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Fig. 1. Curcumin fluorescence intensity in ENU1564 cells treated with different concentrations (0–40 μM) for 24 h. Data represent mean curcumin fluorescence intensity ± S.E.M of at least 30 cells per concentration tested.
treated with 30 μM and 35 μM curcumin for 24 h, respectively (middle panel and right panel). The curcumin IC50 (concentration at which cell number is 50% of control) in ENU1564 cells was previously determined to be between 15 and 20 μM (Ibrahim et al., 2010).
3.2. Curcumin effects on the expression of pro-caspase-3 and Bcl-2 Bcl-2 and procaspase-3 protein levels were significantly decreased in ENU1564 cells after 24 h treatment with 40 μM curcumin (P b 0.0162 and P b 0.0209 respectively), as determined by western blot analysis (Fig. 3). The reduction in procaspase-3 levels indicates that curcumin induced caspase-3 activation leading to procaspase-3 cleavage. In addition, significant increase in apoptosis (as detected with the fluorescent assay using YO-PRO and Hoechst) was observed only at curcumin concentration of 40 μM (data not shown).
3.3. Effects of curcumin on mitochondrial Ca 2+ To identify the role of Ca 2+ in the curcumin-induced apoptosis, we used rhod-2 to measure the mitochondrial Ca 2+ levels following 6 h, 12 h and 24 h of treatment of ENU1564 cells with different concentrations of curcumin. During the first 6 h of curcumin treatment (Fig. 4A), there was no significant difference in mitochondrial Ca + 2 levels between control and treated cells. However, following 12 h (Fig. 4B) and 24 hrs (Fig. 4C) of treatment with curcumin, the mitochondrial Ca 2+ levels in curcumin treated cells were significantly higher than those of control cells at all concentrations tested.
3.4. RU-360 suppressed the mitochondrial Ca 2+ uptake To investigate the role of the mitochondrial uniporter in Ca 2+ uptake, cells were pretreated with the mitochondrial Ca 2+ uniporter inhibitor RU-360 for 1 h prior to curcumin treatment. RU-360 (10 μM) partially inhibited the curcumin-induced significant increase (P b 0.001) in mitochondrial Ca 2+ at curcumin concentrations of 30 μM and 35 μM (Fig. 5). However, pretreatment with 10 μM RU-360 had no effect on mitochondrial Ca 2+ levels in ENU1564 cells treated with the highest curcumin concentration (40 μM) (data not shown).
3.5. SKF-96365 inhibited the mitochondrial Ca 2+ accumulation The store-operated Ca 2+ channel inhibitor SKF-96365 significantly suppressed the curcumin induced effect on mitochondrial Ca 2+ at concentrations equal to or greater than 25 μM following 24 h of treatment (Fig. 6).
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Fig. 2. An example of ENU1564 cells treated with 0 μM (left panel), 30 μM (middle panel) and 35 μM (right panel) curcumin for 24 h. The green fluorescence intensity is indicative of live cells while the red one is indicative of dead cells.
3.6. Effect of curcumin on mitochondrial membrane potential Mitochondrial membrane potential was assessed using the potential sensitive dye JC-1. Curcumin induced a concentrationdependent loss of MMP (Fig. 7A). In cultures treated for 24 h with 35 μM curcumin, depolarization of the inner mitochondrial membrane was complete as indicated by the depolarizing agent CCCP (data not shown). Curcumin-induced depolarization was partially reversed when ENU1564 cells were pretreated with RU-360 (10 μM) for 1 h prior to curcumin treatment (Fig. 7B). 3.7. Effects of curcumin on reactive oxygen species production in ENU1564 cells
demonstrates that cytotoxic levels of curcumin induced apoptosis in murine mammary gland adenocarcinoma (ENU1564) cells through multiple pathways. Curcumin activated the mitochondrial pathway of apoptosis, as evidenced by the increase of mitochondrial Ca 2+ levels and the loss of mitochondrial membrane potential. Curcumin also increased reactive oxygen species production in the cytoplasm, decreased the protein expression of Bcl-2, and increased the activation of caspase-3, as indicated by the reduction of procaspase-3 levels. Other in vitro studies have shown that curcumin induced apoptosis through the mitochondrial dependent pathway in many cancer cell lines, such as human glioblastoma (Karmakar et al., 2006), prostate cancer (Shankar and Srivastava, 2007), lung carcinoma cells (Lin et al., 2008), and cervical carcinoma cells (Singh and Singh, 2009), through interfering with multiple signaling pathways such as
Reactive oxygen species levels in ENU1564 cells were measured using the fluorescent probe CMH2DCFDA. Significant changes in reactive oxygen species production were only noticeable following 24 h of curcumin treatment at the two highest concentrations (35 μM and 40 μM) (Fig. 8). 4. Discussion Curcumin is an intriguing natural drug because it has a diverse range of molecular targets and acts upon numerous biochemical and molecular signaling cascades (Kunnumakkara et al., 2008). This study
Fig. 3. Effects of curcumin on Bcl-2 and Pro-caspase expression in ENU1564 cells. Western blot showing significant decrease in procaspase-3 and Bcl-2 protein expressions following 40 μM curcumin for 24 h with chart indicating densitometric analysis. *Indicates significant difference from control at P b 0.05.
Fig. 4. Mitochondrial Ca2+ measured by the probe rhod-2,AM in ENU1564 cells treated for 6 h (A), 12 h (B) and 24 h (C) and different concentrations of curcumin (0–40 μM). Values represent mean fluorescence intensity +/− S.E.M of 8 wells per concentration. Different symbols above the error bar indicates significant difference in mitochondrial Ca2+ between the concentrations at P b 0.05.
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Fig. 5. Mitochondrial Ca+ 2 uptake measured by the fluorescence intensity of the probe rhod-2 in ENU1564 cells pretreated with RU-360 (black bar) or the solvent control (white bar) for 30 min prior to treatment with different concentration of curcumin (0–35 μM) for 24 h. Values represent mean fluorescence intensity +/−S.E.M of 8 samples per treatment. *Represents significant difference from the corresponding control at P b 0.05. Note that RU-360 has no effect on mitochondrial Ca2+ in cells treated with 40 μM curcumin (data not shown).
activation of caspases (−3, −8, and −9), cleavage of Bid to tBid, increase of Bax:Bcl-2 ratio, release of mitochondrial proteins (cytochrome c, Smac/DIABLO and Omi/HtrA2), translocation of Bax and p53 to mitochondria, production of reactive oxygen species, decrease in mitochondrial membrane potential and upregulation of AIF. Ca2+ has an important role in the activation and execution of cell death processes. The main organelles governing intracellular Ca2+ fluxes are the endoplasmic reticulum (ER) and mitochondria. The ability of mitochondria to acutely sense Ca2+ release from the ER might allow them to act as cellular sentinels of ER-mediated apoptotic signals. There are different ways by which Ca2+ can induce apoptosis such as (a) Ca 2+induced permeability transition pore opening followed by cytochrome c release and activation of caspases, (b) activation of calpains which are potent amplifiers and initiators of death signaling and, (c) activation of apoptosis-linked gene 2 (Roderick and Cook, 2008). Our results have shown that curcumin increased the mitochondrial Ca 2+ level as early as 12 h post treatment at the lowest tested concentration of 25 μM. Moreover, the decrease in mitochondrial membrane potential was not significant starting until cells were treated for 24 h with 25 μM curcumin. This time course indicates that the change in Ca 2+ occurred before the change in mitochondrial membrane potential. Other studies have shown that curcumin induces apoptosis in different cell types and this was associated with an increase in cytoplasmic Ca2+ level and a decrease in mitochondrial membrane potential (An et al., 2009; Lin et al., 2008; Su et al., 2006). In order to confirm the ability of curcumin to increase the mitochondrial Ca2+ level and to decrease the mitochondrial membrane potential, we pretreated cells with RU-360 prior to curcumin treatment, which reversed the action of curcumin at the 30 and 35 μM
concentrations. RU-360 had no effect at the 40 μM concentration of curcumin, possibly due to cells reaching the stage of irreversible damage (apoptosis). Our results indicated that 40 μM curcumin induced apoptosis, this finding was confirmed by the results of RU-360 which did not reverse the action of this concentration. This result is in agreement with a study that used Ruthenium red, an inhibitor of mitochondrial uniporter, to inhibit curcumin-mediated apoptosis via prevention of intracellular Ca2+ depletion (Bae et al., 2003). Depletion of internal Ca 2+ stores activates Ca 2+ influx across the plasma membrane through store-operated calcium influx or capacitative calcium entry (Berridge et al., 2003). This influx of Ca 2+ into the cytoplasm may result in greater Ca 2+ accumulation in mitochondria and potentiate the process of apoptosis induced by curcumin. Other studies have shown that SKF abrogates Ca 2+ influx through store operated Ca 2+ channel and decreases the level of cytosolic Ca 2+ level (Aires et al., 2007; Kolar et al., 2007). We found that SKF blocked the elevation of mitochondrial Ca 2+ level induced by curcumin, thereby implicating potentiation of curcumin-induced apoptosis by the above mentioned process.
Fig. 6. Inhibition of curcumin-induced mitochondrial Ca2+ increase with SKF-96365. Data represent mean fluorescence intensity ± S.E.M of rhod2 in ENU1564 cells at each curcumin concentration tested in the presence (Δ) or absence (▲) of SKF. *Indicates significant difference from the same dose tested in the absence of SKF.
Fig. 8. Effect of different concentrations (0–40 μM) of curcumin on reactive oxygen species production in ENU1564 cells. Values represent mean normalized intensity +/− S.E.M of at least 8 samples per treatment. The symbol * represents significant difference from control at P b 0.05.
Fig. 7. (A) Mitochondrial membrane potential in ENU1564 cells treated for 24 h with different concentrations (0–40 μM) of curcumin. (B) Mitochondrial membrane potential in EN1354 cells pretreated with 10 μM RU-360 (black bar) or solvent control (open bar) for 1 h prior to treatment with different concentrations (0–40 μM) of curcumin. Values represent mean ratio (red fluorescence over green fluorescence of JC-1) +/− S.E.M of 8 samples per treatment. Different symbols above the bar indicates significant difference at P b 0.05.
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Increased mitochondrial Ca2+ and reactive oxygen species generation act synergistically to produce the mitochondrial permeability transition and cell death (Lemasters et al., 2009). Our results have demonstrated that curcumin induced reactive oxygen species production at 35 and 40 μM after 24 h. We can assume that increased mitochondrial Ca2+ level initiated the production of reactive oxygen species, which in turn induced apoptosis at 40 μM concentration of curcumin. These results come side by side with results of Chacon and Acosta (1991) proving that the production of superoxide by respiring rat heart mitochondria was decreased by either chelating extra-mitochondrial Ca2+ with EGTA or by blocking mitochondrial Ca2+ uptake with Ruthenium red. Other studies have shown that curcumin induced apoptosis in several cell lines with induction of intracellular reactive oxygen species production (Atsumi et al., 2006, 2007; Skommer et al., 2006; Tan et al., 2006). Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Therefore, down-regulation of this oncoprotein, represents a possible new way to target clinically aggressive disease (Chanan-Khan, 2005). However, Bcl-2 may promote cell survival by interfering with the activation of the cytochrome c/Apaf-1 pathway through stabilization of the mitochondrial membrane (Yang et al., 1997). Caspase-3 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many key proteins and is expressed in cells as an inactive precursor (Cohen, 1997). As shown from our results, curcumin significantly inhibited Bcl-2 and activated caspase-3 at a curcumin concentration of 40 μM, while no significant effect was observed at lower concentrations. This finding is consistent with the apoptosis data obtained at 40 μM curcumin and in agreement with published data showing similar curcumin results on Bcl-2 and caspase-3 (Bill et al., 2009; Walters et al., 2008). Overall, we can conclude from the results of this study that the elevation of mitochondrial Ca 2+ levels was the leading factor by which curcumin induced apoptosis in ENU1564 cells through mitochondrial pathway of apoptosis. Other mechanisms of apoptosis by order of importance were the generation of reactive oxygen species and finally inhibition of Bcl-2 oncoprotein. These results will provide more insights on the mechanisms of action of curcumin in cells and may facilitate development of agents that will block the mutagenic/ carcinogenic pathways. References Aires, V., Hichami, A., Filomenko, R., Ple, A., Rebe, C., Bettaieb, A., Khan, N.A., 2007. Docosahexaenoic acid induces increases in [Ca2+]i via inositol 1,4,5-triphosphate production and activates protein kinase C gamma and -delta via phosphatidylserine binding site: implication in apoptosis in U937 cells. Mol. Pharmacol. 72, 1545–1556. An, J.B., Ma, J.X., Liu, D.Y., Gao, Y.J., Sheng, M.Y., Wang, H.X., Liu, L.Y., 2009. The effect of curcumin on DNA content, mitochondrial transmembrane potential and calcium of rabbit cultured retinal pigment epithelial cells. Zhonghua Yan Ke Za Zhi 45, 210–215. Atsumi, T., Tonosaki, K., Fujisawa, S., 2006. Induction of early apoptosis and ROSgeneration activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin. Arch. Oral Biol. 51, 913–921. Atsumi, T., Tonosaki, K., Fujisawa, S., 2007. Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase. Anticancer. Res. 27, 363–371. Bae, J.H., Park, J.W., Kwon, T.K., 2003. Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochem. Biophys. Res. Commun. 303, 1073–1079. Bengmark, S., Mesa, M.D., Gil, A., 2009. Plant-derived health: the effects of turmeric and curcuminoids. Nutr. Hosp. 24, 273–281. Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529. Bill, M.A., Bakan, C., Benson Jr., D.M., Fuchs, J., Young, G., Lesinski, G.B., 2009. Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines. Mol. Cancer Ther. 8, 2726–2735. Caroppi, P., Sinibaldi, F., Fiorucci, L., Santucci, R., 2009. Apoptosis and human diseases: mitochondrion damage and lethal role of released cytochrome C as proapoptotic protein. Curr. Med. Chem. 16, 4058–4065. Chacon, E., Acosta, D., 1991. Mitochondrial regulation of superoxide by Ca2+: an alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol. Appl. Pharmacol. 107, 117–128.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway Abdelazim Ibrahim a, Abdelmoniem El-meligy a, Gina Lungu b, Hamdy Fetaih a, Amina Dessouki a, George Stoica b, Rola Barhoumi c,⁎ a b c
Department of Pathology, College of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843–4467, United States Department of Veterinary Integrative BioSciences, Texas A&M University, College Station, TX 77843–4458, United States
a r t i c l e
i n f o
Article history: Received 23 February 2011 Received in revised form 10 June 2011 Accepted 23 June 2011 Available online 8 July 2011 Keywords: Curcumin Apoptosis Cancer Mitochondrial calcium Reactive oxygen species production Mitochondrial uniporter inhibitor
a b s t r a c t Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca 2+ homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca2+ accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca 2+ channel blocker, suppressed the curcumin effect on mitochondrial Ca2+. In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca 2+ is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Interest in the use of medicinal plants to treat human disease has been growing rapidly in the last decade, as they are available, cheap, and thought to be safe. Curcumin, a yellow colored polyphenol, is an active principle of the perennial herb Curcuma longa (commonly known as turmeric). In addition to its extensive use as a food additive, turmeric has been used in traditional medicine for treatment of various inflammatory conditions such as arthritis, colitis and hepatitis. Curcumin has also been shown to suppress multiple signaling pathways and inhibit cell proliferation, invasion, metastasis, and angiogenesis (Bengmark et al., 2009; Kunnumakkara et al., 2008). Apoptosis, the process of programmed cell death, is the desired outcome in the testing of potential anticancer drugs. Many studies have proven that curcumin induces cellular apoptosis (Bae et al., 2003; Ramachandran and You, 1999; Su et al., 2006; Thayyullathil et al., 2008; Wang et al., 2009a, 2009b) through multiple signaling pathways, including cell proliferation pathway, cell survival pathway, caspase activation pathway, tumor suppressor pathway, death
⁎ Corresponding author. Tel.: + 1 979 458 1149; fax: +1 979 847 8981. E-mail address: [email protected] (R. Barhoumi). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.06.048
receptor pathway, mitochondrial pathways, and protein kinase pathway (Ravindran et al., 2009). Mitochondria and intracellular calcium play an important role in apoptosis. In response to the apoptotic stimulus, there is an early increase in cytosolic Ca2+ followed by a delayed increase in mitochondrial Ca2+, which in turn induces the opening of permeability transition pores and disrupts mitochondrial membrane potential (MMP). The collapse of MMP along with release of cytochrome c from mitochondria is followed by activation of caspases, nuclear fragmentation and cell death by apoptosis (Caroppi et al., 2009; Smaili et al., 2003). Cytoplasmic calcium chelators (BAPTA-AM) and inhibitors of mitochondrial calcium uptake (ruthenium red) prevent apoptosis (Kruman and Mattson, 1999). In addition, deregulation of Bcl-2 family members, a frequent feature in human malignant diseases and in some cases the reason behind therapy resistance, can block elevation of mitochondrial calcium and inhibit membrane depolarization in cells exposed to apoptotic stimulus (Frenzel et al., 2009). Reactive oxygen species have also been shown to play an equally important role in the mechanism of apoptosis. Cytochrome c release from mitochondria that triggers caspase activation appears to be largely mediated by reactive oxygen species action (Simon et al., 2000). Specifically, in colon cancer cells curcumin induces cytotoxicity and apoptosis which is mediated by the production of reactive oxygen
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species, an increased Ca 2+ in mitochondria, alteration of mitochondrial membrane potential, and induction of caspase-3 activity. Moreover, curcumin promotes the expression of Bax, cytochrome c, p53 and p21, and inhibits the expression of Bcl-2 (Su et al., 2006). We previously evaluated the effects of curcumin and curcumin formulated with phosphatidylcholine (Meriva) in athymic nude mice inoculated with mammary gland adenocarcinoma (ENU 1564) cells (Ibrahim et al., 2010). In this paper, we report the effects of curcumin on the same cells in vitro to identify the mechanisms by which curcumin induces apoptosis in these cells, the role of MMP and Ca 2+, and how they interact to regulate this process. 2. Materials and methods 2.1. Chemicals and cell line Curcumin, monoclonal mouse IgG antibody against beta-actin, Janus green, and Carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemical Co. (St. Louis, Mo). Curcumin was prepared as a stock solution of 40 mM and stored at −20 ° C until use. The ENU1564 tumor cell line used in this study was developed in our laboratory and originated from an N-ethyl-N nitosourea-induced mammary adenocarcinoma in a female Berlin-Druckrey IV (BD-IV) rat. This highly metastatic cell line (Hall and Stoica, 1994) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) (Invitrogen, Carlsbad, CA, USA). The cells were grown at 37 ° C in a humidified incubator containing 5% CO2 in air. Cells were passaged biweekly and used for experiments when in the exponential growth phase. Live/Dead assay kit, rhod-2 AM, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydro-fluorescein diacetate acetyl ester (CMH2DCFDA), YO-PRO and Hoechst were all purchased from Invitrogen. Rabbit polyclonal IgG anti-caspase-3 and mouse monoclonal IgG anti-Bcl-2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). RU-360 and SKF-96365 were purchased from Calbiochem (LA Jolla, CA). 2.2. Uptake of curcumin and viability assessment in ENU1564 cells ENU1564 cells were plated in a 6-well plate for 1 day followed by treatment with different concentrations of curcumin (0–40 μM) for 24 h. Cells were then washed twice with serum free and phenol red free medium. Fluorescence intensity of curcumin was measured using a Biotek plate reader (Biotek Instruments Inc., Winooski, VT, USA) with an excitation and emission wavelength of 457 nm and 510 nm respectively. Cell count per well was then assessed using the Janus green assay to determine the curcumin fluorescence intensity per cell. The effect of curcumin on cell viability was then assessed using the Live/Dead assay, which distinguishes live cells by the presence of ubiquitous intracellular esterase activity determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells and its uniform green fluorescence can be measured with an excitation wavelength of 488 nm and an emission wavelength of 515 nm. The kit also measures dead cells using ethidium homodimer-1 (EthD-1). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids. It can be measured with an excitation wavelength of 488 nm and an emission wavelength of 635 nm. To perform the live/dead assay, cells were loaded with 10 μM calcein-AM and 5 μM EthD-1 for 30 min and then washed. Selected areas from each plate were scanned and images were collected and saved on a BioRad radiance 2000MP laser scanning microscope (Bio-Rad, UK). To quantitate apoptosis in ENU1564 cells following curcumin treatments, cells were loaded with Hoechst 33342 and YO-PRO. YO-
PRO dye enters apoptotic cells and shows green fluorescence. Bluefluorescent Hoechst 33342 brightly stains the condensed chromatin of apoptotic cells and more dimly stains the normal chromatin of living cells. Cells were stained with these dyes according to Invitrogen protocol. Fluorescence emission at 460 nm and 530 nm were measured using excitation of 400 nm for Hoechst and 488 nm for YO-PRO. 2.3. Western blot analysis of pro-caspase3 and Bcl-2 The cells were washed twice with cold Hank's solution and lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and supplemented with a mixture of protease inhibitors (Sigma, St. Louis, MO, USA). The lysates were cleared by centrifugation at 13,000 g at 4 °C for 30 min and the supernatant kept frozen at −80 °C. The protein content of the lysates was determined using Bradford Assay (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard. Proteins (15–30 μg) were separated by 9–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Membranes were incubated for 1 h in blocking buffer (20 mM Tris-HCl-buffered saline containing 5% nonfat milk powder and 0.1% Tween-20) at room temperature, then probed with appropriate antibodies in blocking buffer or blocking buffer including 5% bovine serum albumin instead of 5% nonfat milk overnight at 4 °C. Blots were incubated at 4 °C overnight with anti-BCL-2 (1:200), antipro-caspase-3 (1:200). The blots were washed extensively and then incubated for 1 h with a 1:5,000 dilution of anti-primary antibody purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). After additional washes, the blots were incubated with chemiluminescent substrate, according to the kit instructions (Super Signal West Pico, Pierce, Rockford, IL). 2.4. Effects of curcumin on store operated channels and mitochondrial Ca 2+ in ENU1564 cells The fluorescent probe rhod-2 AM was used to determine the effects of curcumin on mitochondrial calcium. Briefly, ENU1564 cells were seeded into 96 well plates in 8 replicates per treatment. The cells were incubated for 24 h at 37 °C and 5% CO2. Different concentrations of curcumin (0, 25, 30, 35, 40 μM) were added to the cells. Cells were then incubated for 6, 12, and 24 h. Following treatments, cells were washed twice with serum- and phenol red- free medium and labeled with 3 μM rhod-2 AM for 1 h at 37 °C. Cells were then washed twice, and the mitochondrial Ca 2+ was measured using excitation and emission wavelengths of 540 and 590 nm respectively on the Biotek Synergy 4 plate reader. To identify the role of the mitochondrial uniporter in curcumin induced toxicity, cells were incubated with RU-360 (10 μM), an inhibitor of the mitochondrial uniporter, for 30 min prior to curcumin treatment (Nutt et al., 2002). Cells were then washed and treated with different concentrations of curcumin for 24 h. For quantification of rhod-2 fluorescence, each concentration of curcumin was compared to its own control and all rhod-2 fluorescence intensity values were corrected to the cell number per well determined using the Janus green assay as described below. To investigate the role of store operated Ca 2+ channels and extracellular Ca 2+ influx induced by curcumin on the mitochondrial Ca 2+, ENU1564 cells were co-incubated with 10 μM SKF-96365 and curcumin (0–40 μM), prior to mitochondrial Ca 2+ measurements. 2.5. Analysis of mitochondrial membrane potential (MMP) The effects of curcumin on the mitochondrial membrane potential were measured using JC-1. JC-1 is a cationic dye that exhibits potentialdependent accumulation in mitochondria and is characterized by an excitation wavelength of 490 nm and two emission peaks at 539 nm and 597 nm corresponding to monomer (green) and aggregate (red)
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forms of the dye respectively. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (Di Lisa et al., 1995). In a 96 well plate, tumor cells were treated with different concentrations of curcumin (0, 25, 30, 35, 40 μM). Following 24 h of treatment, cells were washed and loaded with 5 μg/ml of JC-1 for 30 min. To determine how much depolarization is occurring in ENU1564 cells due to curcumin treatment, results with JC-1 were compared to a calibration curve obtained with different concentrations of the uncoupling agent CCCP for different time intervals (Zamzami et al., 1995). In addition, the curcumin effects on MMP were analyzed in ENU1564 cells treated with 10 μM of the mitochondrial uniporter inhibitor RU-360 for 30 min to 1 h prior to curcumin treatment. 2.6. Assessment of reactive oxygen species generation Generation of reactive oxygen species in ENU1564 cells, following treatment with different concentrations of curcumin, was assessed using the fluorescent probe CMH2DCFDA (Hempel et al., 1999). Curcumin treated cells were incubated with 5 μM CMH2DCFDA for 30 min at 37 °C. Cells were then washed, excited at 488 nm wavelength and fluorescence emission per well was measured at 515 nm wavelength using a Biotek Synergy 4 Plate Reader. Reactive oxygen species per cell were computed by dividing the fluorescence intensity measured by the number of cells per well determined using the Janus green assay (described below). 2.7. Cell counts For cell counting in wells, cultures were washed twice with PBS and fixed with methanol for 30 min at room temperature. Methanol was completely removed and 1 mg/ml janus green in PBS was added to the cultures for 3 min. Following removal of Janus green, cultures were washed twice with PBS and 100 μl of 50% methanol was added to each well. Cell counts were then determined with a Biotek synergy 4 plate reader using an absorbance of 654 nm (Raspotnig et al., 1999). 2.8. Statistical analysis Quantification of pro-caspase 3 and Bcl-2 bands was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http:// rsb.info.nih.gov/nih-image). Data were presented as mean ± S.D., and statistical comparisons were made using GraphPad Prism version 4.00 for Windows, (GraphPad Software, San Diego, California, USA, www. graphpad.com). A P-value less than 0.05 was considered statistically significant. Mitochondrial Ca 2+ was presented as mean fluorescence intensity ± S.E.M and mitochondrial membrane potential was presented as ratio (red/green) ± S.E.M. For comparison of response with different concentrations of curcumin, one way ANOVA was used followed by Tukey's multiple comparison test. For experiments involving RU-360, two-way ANOVA was used followed by Bonferroni test. Differences between treatments were considered significant at P b 0.05. 3. Results 3.1. Curcumin decreased the viability of ENU1564 cells The uptake of different concentrations of curcumin by ENU1564 cells following 24 h treatments was recorded, as shown in Fig. 1. Using the live/dead assay to evaluate the toxicity of curcumin, the results showed that the number of dead cells, as indicated by the EthD-1 red fluorescence, increases with increasing curcumin concentrations (0–40 μM). Fig. 2 shows the live (green fluorescence) and dead (red fluorescence) cells in a control culture (left panel) and in cultures
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Fig. 1. Curcumin fluorescence intensity in ENU1564 cells treated with different concentrations (0–40 μM) for 24 h. Data represent mean curcumin fluorescence intensity ± S.E.M of at least 30 cells per concentration tested.
treated with 30 μM and 35 μM curcumin for 24 h, respectively (middle panel and right panel). The curcumin IC50 (concentration at which cell number is 50% of control) in ENU1564 cells was previously determined to be between 15 and 20 μM (Ibrahim et al., 2010).
3.2. Curcumin effects on the expression of pro-caspase-3 and Bcl-2 Bcl-2 and procaspase-3 protein levels were significantly decreased in ENU1564 cells after 24 h treatment with 40 μM curcumin (P b 0.0162 and P b 0.0209 respectively), as determined by western blot analysis (Fig. 3). The reduction in procaspase-3 levels indicates that curcumin induced caspase-3 activation leading to procaspase-3 cleavage. In addition, significant increase in apoptosis (as detected with the fluorescent assay using YO-PRO and Hoechst) was observed only at curcumin concentration of 40 μM (data not shown).
3.3. Effects of curcumin on mitochondrial Ca 2+ To identify the role of Ca 2+ in the curcumin-induced apoptosis, we used rhod-2 to measure the mitochondrial Ca 2+ levels following 6 h, 12 h and 24 h of treatment of ENU1564 cells with different concentrations of curcumin. During the first 6 h of curcumin treatment (Fig. 4A), there was no significant difference in mitochondrial Ca + 2 levels between control and treated cells. However, following 12 h (Fig. 4B) and 24 hrs (Fig. 4C) of treatment with curcumin, the mitochondrial Ca 2+ levels in curcumin treated cells were significantly higher than those of control cells at all concentrations tested.
3.4. RU-360 suppressed the mitochondrial Ca 2+ uptake To investigate the role of the mitochondrial uniporter in Ca 2+ uptake, cells were pretreated with the mitochondrial Ca 2+ uniporter inhibitor RU-360 for 1 h prior to curcumin treatment. RU-360 (10 μM) partially inhibited the curcumin-induced significant increase (P b 0.001) in mitochondrial Ca 2+ at curcumin concentrations of 30 μM and 35 μM (Fig. 5). However, pretreatment with 10 μM RU-360 had no effect on mitochondrial Ca 2+ levels in ENU1564 cells treated with the highest curcumin concentration (40 μM) (data not shown).
3.5. SKF-96365 inhibited the mitochondrial Ca 2+ accumulation The store-operated Ca 2+ channel inhibitor SKF-96365 significantly suppressed the curcumin induced effect on mitochondrial Ca 2+ at concentrations equal to or greater than 25 μM following 24 h of treatment (Fig. 6).
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Fig. 2. An example of ENU1564 cells treated with 0 μM (left panel), 30 μM (middle panel) and 35 μM (right panel) curcumin for 24 h. The green fluorescence intensity is indicative of live cells while the red one is indicative of dead cells.
3.6. Effect of curcumin on mitochondrial membrane potential Mitochondrial membrane potential was assessed using the potential sensitive dye JC-1. Curcumin induced a concentrationdependent loss of MMP (Fig. 7A). In cultures treated for 24 h with 35 μM curcumin, depolarization of the inner mitochondrial membrane was complete as indicated by the depolarizing agent CCCP (data not shown). Curcumin-induced depolarization was partially reversed when ENU1564 cells were pretreated with RU-360 (10 μM) for 1 h prior to curcumin treatment (Fig. 7B). 3.7. Effects of curcumin on reactive oxygen species production in ENU1564 cells
demonstrates that cytotoxic levels of curcumin induced apoptosis in murine mammary gland adenocarcinoma (ENU1564) cells through multiple pathways. Curcumin activated the mitochondrial pathway of apoptosis, as evidenced by the increase of mitochondrial Ca 2+ levels and the loss of mitochondrial membrane potential. Curcumin also increased reactive oxygen species production in the cytoplasm, decreased the protein expression of Bcl-2, and increased the activation of caspase-3, as indicated by the reduction of procaspase-3 levels. Other in vitro studies have shown that curcumin induced apoptosis through the mitochondrial dependent pathway in many cancer cell lines, such as human glioblastoma (Karmakar et al., 2006), prostate cancer (Shankar and Srivastava, 2007), lung carcinoma cells (Lin et al., 2008), and cervical carcinoma cells (Singh and Singh, 2009), through interfering with multiple signaling pathways such as
Reactive oxygen species levels in ENU1564 cells were measured using the fluorescent probe CMH2DCFDA. Significant changes in reactive oxygen species production were only noticeable following 24 h of curcumin treatment at the two highest concentrations (35 μM and 40 μM) (Fig. 8). 4. Discussion Curcumin is an intriguing natural drug because it has a diverse range of molecular targets and acts upon numerous biochemical and molecular signaling cascades (Kunnumakkara et al., 2008). This study
Fig. 3. Effects of curcumin on Bcl-2 and Pro-caspase expression in ENU1564 cells. Western blot showing significant decrease in procaspase-3 and Bcl-2 protein expressions following 40 μM curcumin for 24 h with chart indicating densitometric analysis. *Indicates significant difference from control at P b 0.05.
Fig. 4. Mitochondrial Ca2+ measured by the probe rhod-2,AM in ENU1564 cells treated for 6 h (A), 12 h (B) and 24 h (C) and different concentrations of curcumin (0–40 μM). Values represent mean fluorescence intensity +/− S.E.M of 8 wells per concentration. Different symbols above the error bar indicates significant difference in mitochondrial Ca2+ between the concentrations at P b 0.05.
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Fig. 5. Mitochondrial Ca+ 2 uptake measured by the fluorescence intensity of the probe rhod-2 in ENU1564 cells pretreated with RU-360 (black bar) or the solvent control (white bar) for 30 min prior to treatment with different concentration of curcumin (0–35 μM) for 24 h. Values represent mean fluorescence intensity +/−S.E.M of 8 samples per treatment. *Represents significant difference from the corresponding control at P b 0.05. Note that RU-360 has no effect on mitochondrial Ca2+ in cells treated with 40 μM curcumin (data not shown).
activation of caspases (−3, −8, and −9), cleavage of Bid to tBid, increase of Bax:Bcl-2 ratio, release of mitochondrial proteins (cytochrome c, Smac/DIABLO and Omi/HtrA2), translocation of Bax and p53 to mitochondria, production of reactive oxygen species, decrease in mitochondrial membrane potential and upregulation of AIF. Ca2+ has an important role in the activation and execution of cell death processes. The main organelles governing intracellular Ca2+ fluxes are the endoplasmic reticulum (ER) and mitochondria. The ability of mitochondria to acutely sense Ca2+ release from the ER might allow them to act as cellular sentinels of ER-mediated apoptotic signals. There are different ways by which Ca2+ can induce apoptosis such as (a) Ca 2+induced permeability transition pore opening followed by cytochrome c release and activation of caspases, (b) activation of calpains which are potent amplifiers and initiators of death signaling and, (c) activation of apoptosis-linked gene 2 (Roderick and Cook, 2008). Our results have shown that curcumin increased the mitochondrial Ca 2+ level as early as 12 h post treatment at the lowest tested concentration of 25 μM. Moreover, the decrease in mitochondrial membrane potential was not significant starting until cells were treated for 24 h with 25 μM curcumin. This time course indicates that the change in Ca 2+ occurred before the change in mitochondrial membrane potential. Other studies have shown that curcumin induces apoptosis in different cell types and this was associated with an increase in cytoplasmic Ca2+ level and a decrease in mitochondrial membrane potential (An et al., 2009; Lin et al., 2008; Su et al., 2006). In order to confirm the ability of curcumin to increase the mitochondrial Ca2+ level and to decrease the mitochondrial membrane potential, we pretreated cells with RU-360 prior to curcumin treatment, which reversed the action of curcumin at the 30 and 35 μM
concentrations. RU-360 had no effect at the 40 μM concentration of curcumin, possibly due to cells reaching the stage of irreversible damage (apoptosis). Our results indicated that 40 μM curcumin induced apoptosis, this finding was confirmed by the results of RU-360 which did not reverse the action of this concentration. This result is in agreement with a study that used Ruthenium red, an inhibitor of mitochondrial uniporter, to inhibit curcumin-mediated apoptosis via prevention of intracellular Ca2+ depletion (Bae et al., 2003). Depletion of internal Ca 2+ stores activates Ca 2+ influx across the plasma membrane through store-operated calcium influx or capacitative calcium entry (Berridge et al., 2003). This influx of Ca 2+ into the cytoplasm may result in greater Ca 2+ accumulation in mitochondria and potentiate the process of apoptosis induced by curcumin. Other studies have shown that SKF abrogates Ca 2+ influx through store operated Ca 2+ channel and decreases the level of cytosolic Ca 2+ level (Aires et al., 2007; Kolar et al., 2007). We found that SKF blocked the elevation of mitochondrial Ca 2+ level induced by curcumin, thereby implicating potentiation of curcumin-induced apoptosis by the above mentioned process.
Fig. 6. Inhibition of curcumin-induced mitochondrial Ca2+ increase with SKF-96365. Data represent mean fluorescence intensity ± S.E.M of rhod2 in ENU1564 cells at each curcumin concentration tested in the presence (Δ) or absence (▲) of SKF. *Indicates significant difference from the same dose tested in the absence of SKF.
Fig. 8. Effect of different concentrations (0–40 μM) of curcumin on reactive oxygen species production in ENU1564 cells. Values represent mean normalized intensity +/− S.E.M of at least 8 samples per treatment. The symbol * represents significant difference from control at P b 0.05.
Fig. 7. (A) Mitochondrial membrane potential in ENU1564 cells treated for 24 h with different concentrations (0–40 μM) of curcumin. (B) Mitochondrial membrane potential in EN1354 cells pretreated with 10 μM RU-360 (black bar) or solvent control (open bar) for 1 h prior to treatment with different concentrations (0–40 μM) of curcumin. Values represent mean ratio (red fluorescence over green fluorescence of JC-1) +/− S.E.M of 8 samples per treatment. Different symbols above the bar indicates significant difference at P b 0.05.
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Increased mitochondrial Ca2+ and reactive oxygen species generation act synergistically to produce the mitochondrial permeability transition and cell death (Lemasters et al., 2009). Our results have demonstrated that curcumin induced reactive oxygen species production at 35 and 40 μM after 24 h. We can assume that increased mitochondrial Ca2+ level initiated the production of reactive oxygen species, which in turn induced apoptosis at 40 μM concentration of curcumin. These results come side by side with results of Chacon and Acosta (1991) proving that the production of superoxide by respiring rat heart mitochondria was decreased by either chelating extra-mitochondrial Ca2+ with EGTA or by blocking mitochondrial Ca2+ uptake with Ruthenium red. Other studies have shown that curcumin induced apoptosis in several cell lines with induction of intracellular reactive oxygen species production (Atsumi et al., 2006, 2007; Skommer et al., 2006; Tan et al., 2006). Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Therefore, down-regulation of this oncoprotein, represents a possible new way to target clinically aggressive disease (Chanan-Khan, 2005). However, Bcl-2 may promote cell survival by interfering with the activation of the cytochrome c/Apaf-1 pathway through stabilization of the mitochondrial membrane (Yang et al., 1997). Caspase-3 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many key proteins and is expressed in cells as an inactive precursor (Cohen, 1997). As shown from our results, curcumin significantly inhibited Bcl-2 and activated caspase-3 at a curcumin concentration of 40 μM, while no significant effect was observed at lower concentrations. This finding is consistent with the apoptosis data obtained at 40 μM curcumin and in agreement with published data showing similar curcumin results on Bcl-2 and caspase-3 (Bill et al., 2009; Walters et al., 2008). Overall, we can conclude from the results of this study that the elevation of mitochondrial Ca 2+ levels was the leading factor by which curcumin induced apoptosis in ENU1564 cells through mitochondrial pathway of apoptosis. Other mechanisms of apoptosis by order of importance were the generation of reactive oxygen species and finally inhibition of Bcl-2 oncoprotein. These results will provide more insights on the mechanisms of action of curcumin in cells and may facilitate development of agents that will block the mutagenic/ carcinogenic pathways. References Aires, V., Hichami, A., Filomenko, R., Ple, A., Rebe, C., Bettaieb, A., Khan, N.A., 2007. Docosahexaenoic acid induces increases in [Ca2+]i via inositol 1,4,5-triphosphate production and activates protein kinase C gamma and -delta via phosphatidylserine binding site: implication in apoptosis in U937 cells. Mol. Pharmacol. 72, 1545–1556. An, J.B., Ma, J.X., Liu, D.Y., Gao, Y.J., Sheng, M.Y., Wang, H.X., Liu, L.Y., 2009. The effect of curcumin on DNA content, mitochondrial transmembrane potential and calcium of rabbit cultured retinal pigment epithelial cells. Zhonghua Yan Ke Za Zhi 45, 210–215. Atsumi, T., Tonosaki, K., Fujisawa, S., 2006. Induction of early apoptosis and ROSgeneration activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin. Arch. Oral Biol. 51, 913–921. Atsumi, T., Tonosaki, K., Fujisawa, S., 2007. Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase. Anticancer. Res. 27, 363–371. Bae, J.H., Park, J.W., Kwon, T.K., 2003. Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochem. Biophys. Res. Commun. 303, 1073–1079. Bengmark, S., Mesa, M.D., Gil, A., 2009. Plant-derived health: the effects of turmeric and curcuminoids. Nutr. Hosp. 24, 273–281. Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529. Bill, M.A., Bakan, C., Benson Jr., D.M., Fuchs, J., Young, G., Lesinski, G.B., 2009. Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines. Mol. Cancer Ther. 8, 2726–2735. Caroppi, P., Sinibaldi, F., Fiorucci, L., Santucci, R., 2009. Apoptosis and human diseases: mitochondrion damage and lethal role of released cytochrome C as proapoptotic protein. Curr. Med. Chem. 16, 4058–4065. Chacon, E., Acosta, D., 1991. Mitochondrial regulation of superoxide by Ca2+: an alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol. Appl. Pharmacol. 107, 117–128.
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