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</svg>-Dependent Pathway

Wang, Wei-Hsun; Chiang, I-Tsang; Ding, Kuke; Chung, Jing-Gung; Lin, Wuu-Jyh; Lin, Song-Shei; Hwang, Jeng-Jong

doi:https://doi.org/10.1155/2012/512907

Evidence-Based Complementary and Alternative Medicine

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Research Article | Open Access

Volume 2012 | Article ID 512907 | https://doi.org/10.1155/2012/512907

Curcumin-Induced Apoptosis in Human Hepatocellular Carcinoma J5 Cells: Critical Role of -Dependent Pathway

Wei-Hsun Wang,^1,2I-Tsang Chiang,¹Kuke Ding,³Jing-Gung Chung,⁴Wuu-Jyh Lin,⁵Song-Shei Lin,⁶and Jeng-Jong Hwang¹

Academic Editor: Cheppail Ramachandran

Received11 Sept 2011

Revised01 Dec 2011

Accepted18 Dec 2011

Published01 Apr 2012

Abstract

The antitumor effects of curcumin, a natural biologically active compound extracted from rhizomes of Curcuma longa, have been studied in many cancer cell types including human hepatocellular carcinoma (HCC). Here, we investigated the effects of Ca²⁺ on curcumin-induced apoptosis in human HCC J5 cells. The abrogation of mitochondrial membrane potential (Δ), the increase of reactive oxygen species (ROS) production, and calcium release were demonstrated with flow cytometry as early as 15 minutes after curcumin treatment. In addition, an increase level of cytochrome c in the cytoplasm which led to DNA fragmentation was observed. To verify the role of Ca²⁺ in curcumin-induced apoptosis, 1,2-bis(o-aminophenoxy)ethane-N,N,,-tetraacetic acid (BAPTA), an intracellular calcium chelator, was applied. Cell viability was increased, but Δ, ROS production, activation of caspase 3, and cell death were decreased in J5 cells pretreated with BAPTA for 2 h followed by the treatment of 25 μM curcumin. These results suggest that the curcumin-induced apoptosis in human HCC J5 cells is via mitochondria-dependent pathway and is closely related to the level of intracellular accumulation of calcium.

1. Introduction

Human HCC treated with chemotherapy often turned out with poor prognosis [1, 2]. Curcumin, one of phytochemical compounds, has been shown with chemopreventive and chemotherapeutic properties against tumors in animal models and clinical trials [3–5]. Curcumin induces the apoptosis of tumor cells through mitochondria-dependant pathways, including the release of cytochrome c, changes in electron transport, and loss of mitochondrial transmembrane potential [6]. Curcumin can stimulate intracellular Ca²⁺ uptake into the mitochondria [7], resulting in the stimulation of oxidative phosphorylation, transmission, and amplification of the apoptotic signal via the suppression of mitochondria membrane potential and the release of cytochrome c [8]. Apoptosis induced by curcumin in human HepG2 cells has been shown through mitochondrial hyperpolarization and DNA damage [9]. Mitochondria are the moderator of intracellular Ca²⁺ dynamics and transport through a complex system consisting of two modes of influx and efflux [7]. Oxidative phosphorylation can be stimulated by the accumulation of Ca²⁺ in the mitochondrial matrix, then transmit and amplify the apoptotic signal [8]. Apoptosis induced by curcumin also has been reported through the prevention of intracellular Ca²⁺ depletion and the release of cytochrome c in mouse melanoma cells [10]. We hypothesize that curcumin-induced Ca²⁺ release will result in mitochondrial Ca²⁺ overuptake to affect mitochondria membrane potential stability. To prove this, we choose BAPTA, an intracellular Ca²⁺ chelator, as the inhibitor for mitochondrial Ca²⁺ uptake [11]. However, previous study indicates that curcumin-induced apoptosis is through ER stress dependent pathway, that is, GADD153 transcription activation [12]. In this study, we demonstrated that curcumin-induced apoptosis in human HCC J5 cells is via Ca²⁺-regulated mitochondria-dependent pathway.

2. Materials and Methods

2.1. Cell Culture

The HCC J5 cell line was obtained from the Cell Culture Center of the National Taiwan University (Taipei, Taiwan). Cells were cultured with DMEM supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10% fetal bovine serum, and 2% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin in a 5% CO₂ humidified incubator).

2.2. Morphological Study and Cell Viability

The J5 cells were cultured in 12-well plates at a density of 2 × 10⁵ cells/well for 24 h, then treated with various concentrations of curcumin (0, 10, 15, 20, 25, and 50 μM in 0.1% DMSO) for different time periods. Trypan blue exclusion was used to the cell viability as previously described [13]. In short, approximately 10 μL of cell suspensions in PBS were mixed with 40 μL of trypan blue. The numbers of stained (dead cells) and unstained cells (live cells) were counted under a light microscope. At least, 5000 cells were counted. The cell viability is calculated using the following formula:

2.3. Comet Assay

2 × 10⁵ J5 cells/well were grown in 12-well plates and treated with curcumin at 0, 25, and 50 μM for 24 h, then examined for DNA damage using Comet assay. Cells were harvested and mixed with low melting point agarose. The mixture was then placed in the solid normal melting point agarose on the slide covered with coverslip. The coverslip was removed after the agarose was gelled at 4°C. The slide was transferred to the lysis buffer at 4°C for 1 h before putting in alkaline buffer for electrophoresis (25 V, 300 mA). The slide was washed with neutralized buffer and stained with PI after electrophoresis [13].

2.4. DNA Fragmentation

0⁶ J5 cells/well were grown in 6-well plates and treated with 25 μM curcumin for 12, 24, 36, and 48 h. The fragmented DNA was extracted using a cell genomic DNA purification kit (Genemark). The DNA extracted procedures followed the protocols provided by the manufacture. The DNA fragmentation was assayed with 1.5% agarose gel electrophoresis.

2.5. Caspase-3 Activity Assay

2 × 10⁵ J5 cells/well were cultured in 12-well plates and treated with 25 μM curcumin for various time periods. Cells were harvested in a 15-mL centrifuge tube by centrifugation. 50 μL of 10 μM PhiPhilux solution, a substrate for caspase-3, was added to each well and incubated at 37°C for 1 h. Cells were then washed once with 1 mL of ice-cold PBS and resuspended in fresh 1 mL PBS. Caspase-3 activity was analyzed by flow cytometry (Becton-Dickinson, CA, USA) equipped with an argon ion laser at 488 nm wavelength [13]. In addition, J5 cells were pretreated with 10 μM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), a calcium chelator, for 2 h, then were assayed for caspase-3 activity as described in the above.

2.6. Detection of Reactive Oxygen Species (ROS)

2 × 10⁵ J5 cells/well in 12-well plates were incubated with 25 μM curcumin for different time periods to detect the changes of ROS. Cells were harvested and washed twice, resuspended in 500 μL of 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and incubated at 37°C for 30 min, then analyzed by flow cytometry [13].

2.7. Detection of Mitochondrial Membrane Potential (ΔΨ_m)

2 × 10⁵ J5 cells/well in 12-well plates were incubated with 25 μM curcumin for different time course to determine the changes in ΔΨ_m. Cells were harvested and washed twice, resuspended in 500 μL of 4 μM DiOC₆, and incubated at 37°C for 30 min, then analyzed by flow cytometry [13].

2.8. Cell Viability, ROS Production, ΔΨ_m Levels in J5 cells Pre-Treated with BAPTA

2 × 10⁵ J5 cells/well in 12-well plates were pre-treated with 100 μM BAPTA for 2 h, then treated with 25 μM curcumin for 24 h. Cells were harvested and washed twice, half of cells were analyzed for cell viability with PI staining, the rest was resuspended in 4 μM DiOC₆ and 10 μM DCFH-DA before incubated at 37°C for 30 min, then analyzed by flow cytometry.

2.9. Determination of Ca²⁺ Concentration

2 × 10⁵ J5 cells/well in 12-well plates were incubated with 25 μM curcumin for various time intervals to determine the Ca²⁺ levels. Cells were harvested and washed twice, resuspended in 3 μg/mL Indo 1/AM, incubated at 37°C for 30 min, and analyzed by flow cytometry.

2.10. Western Blotting

2 × 10⁵ J5 cells/well in 12-well plates were treated with 25 μM curcumin for 0, 6, 12, 24, and 48 h. The level of cytochrome c in the cytosol was isolated according to the manufacturer’s protocol (A cytosol/nuclear extraction kit purchased from Chemicon International, Temecula, CA, USA). The total proteins of cells were extracted with cell lysis buffer (50 mM Tris-HCL pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM PMSF), and 40 μg of protein extract was separated by 10% SDS-PAGE, then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), blocked with 5% nonfat milk in TBSTween buffer (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween20) for 1 hour at room temperature, and incubated with the appropriate antibody overnight at 4°C, then incubated with horseradish peroxidase conjugated secondary antibody for 30 min at room temperature. The bound antibody was detected with peroxidase-conjugated anti-rabbit antibody (1 : 10000) or anti-mouse antibody (1 : 10000) followed by chemiluminescence (ECL System) and exposed by autoradiography. The following primary antibodies except cytochrome c (1 : 500) (Oncogene Research Products): β-actin (1 : 10000), Bcl-2 (1 : 1000), Bcl-xl (1 : 1000), Fas (1 : 1000), caspase-8 (1 : 1000), caspase-12 (1 : 1000), and catalase (1 : 1000) were purchased from Upstate, Millipore.

2.11. Statistics

Student’s t-test was used to evaluate the significance or values between groups (*, **). Standard errors of mean values were depicted as error bars in all figures.

3. Results

3.1. Morphological Study and Cell Viability

The morphology of J5 cells induced by curcumin was remained unchanged, but the apoptotic bodies could be observed (Figure 1(a)), and increased with times. Figure 1(b) shows the viability of J5 cells are decreased with the increase of curcumin concentration (10–50 μM).

(a)

(b)

3.2. Ca²⁺ Production, Mitochondria Membrane Potential (ΔΨ_m), and Production of Reactive Oxygen Species (ROS) Affected by Curcumin in J5 Cells

Figure 2(a) showed that Ca²⁺ production was significantly enhanced from 15 min up to 720 min by 25 μM curcumin treatment, while the mitochondria membrane potential (ΔΨ_m) was significantly decreased (Figure 2(b)) as compared with that of the control. Reactive oxygen species (ROS) was also significantly increased and reached the highest levels at 15–60 min after 25 μM curcumin treatment (Figure 2(c)).

(a)

(b)

(c)

Figure 2

Flow cytometric analysis of Ca²⁺ concentration, mitochondrial membrane potential (ΔΨ_m), and reactive oxygen species (ROS) in J5 cells treated with curcumin. 5 × 10⁵ J5 cells/well in 12-well plates were incubated with 25 μM curcumin for different time periods. (a) The release of Ca²⁺ as a function of time. (b) Mitochondrial membrane potential (ΔΨ_m) assay. (c) Changes of ROS production as a function of time. Each data point is the mean ± SE of three experiments. * and ** are significantly different from that of the control.

3.3. The Release of Cytochrome c and Apoptotic-Associated Proteins Affected by Curcumin in J5 Cells

To characterize the molecular mechanisms of curcumin-induced apoptosis in J5 cells, the expressions of apoptotic-associated proteins were examined with Western blotting. Figure 3(a) showed that cytochrome c was released from the mitochondria to the cytosol in J5 cells treated with 25 μM curcumin for different time periods (6–48 h). On the other hand, the protein levels of Bcl-2, Bcl-xL, and Fas were decreased. Both caspase-12 and catalase were increased after curcumin treatment for 6 and 12 h, but decreased for 24 and 36 h, then increased again for 48 h. Procaspase-8 were not affected by curcumin treatment.

(a)

(b)

3.4. DNA Damage and Fragmentation Caused by Curcumin in J5 Cells

DAPI staining was used to detect the DNA damage in J5 cells treated with curcumin. Figure 4(a) showed that the nuclei of control cells were round and smaller as compared with the condensed and larger nuclei of cells exposed to 25 and 50 μM curcumin for 24 h. The DNA damage induced by curcumin was in a dose-dependent manner. The Comet assay also showed the similar results. The 50 μM curcumin treatment showed a longer DNA migration smear (Figure 4(b)), indicating that more DNA was damaged in the cells. DNA fragmentations were found in J5 cells after12, 24, 36, and 48 h of continuous exposure to 25 μM curcumin as shown in Figure 4(c). The induction of DNA fragmentation by curcumin was in a time-dependent manner.

(a)

(b)

(c)

3.5. Effects of Calcium Chelator BAPTA on Cell Viability, ΔΨ_m, ROS Production, and Caspase-3 Activity Induced by Curcumin in J5 Cells

J5 cells were pretreated with 100 μM BAPTA for 2 h, followed by incubation with 25 μM curcumin for different time periods. Cell viability, ΔΨ_m, ROS, and caspase-3 activity were analyzed by flow cytometry. Figure 5(a) showed that BAPTA could rescue the cell death from curcumin treatment. The recovery of mitochondria membrane potential ΔΨ_m and the inhibition of ROS by BAPTA were shown in Figures 5(b) and 5(c), respectively. In addition, caspase-3 activity increased by 25 μM curcumin was inhibited by BAPTA.

(a)

(b)

(c)

(d)

4. Discussion

We have demonstrated that DNA damage and endoplasmic reticulum (ER) stress-mediated curcumin-induced cell cycle arrest and apoptosis are through the activation of caspases, and mitochondria-dependent pathways in A549 cells [13]; here we further show the similar finding in human hepatocellular carcinoma J5 cells. Mitochondrial dysfunction associated with apoptosis is characterized with the loss of mitochondrial membrane potential (ΔΨ_m), permeability transition, and the release of cytochrome c from the mitochondria into the cytosol [14]. We also show that curcumin induces apoptosis in human HCC J5 cells via mitochondrial-dependent pathway with the suppression of both mitochondria membrane potential (ΔΨ_m) and the induction of cytochrome c release; nevertheless, the ROS production is induced and the Ca²⁺ in cytoplasm is accumulated. Other than mitochondrial dysfunction, the mechanisms responsible for curcumin-induced apoptosis in different cancer cell types may also involve the activation of caspases, and the inhibition of antiapoptotic Bcl-2 family proteins [15–17]. We also found that curcumin decreased the protein levels of Bcl-2 and Bcl-xL in this study. Dröge et al. reported that high levels of ROS induce DNA damage, and result in the cell death [18]. Our result indicates that ROS production in J5 cells with the highest levels at 15–60 min after 25 μM curcumin treatment. Both superoxide dismutase (SOD) and catalase of ROS scavenger reduced ROS production [19]. We also found that curcumin increased protein levels of catalase after curcumin treatment for 6 and 12 h in J5 cells. ΔΨ_m depletion, cytochrome c release, ROS production, and DNA damage caused by curcumin all have contribution on the cell death. However, neither of the aforementioned results, in which multiple related mechanisms of curcumin-induced apoptosis was revealed, indicate the key molecule with potential to steer the pharmacologic effect of curcumin.

Intracellular-free calcium ([Ca²⁺]_i) is a universal signaling molecule regulating many cellular functions including apoptosis. In addition, Ca²⁺-dependent processes are closely related with the mainstream apoptosis executioners, that is, caspases [20]. It is also shown to activate and modulate the execution of a nonapoptotic cell death in C. elegans [21]. Both the overload and the depletion of endoplasmic reticulum Ca²⁺ pool result in the induction of ER stress, and further initiate the apoptotic pathway via activation of procaspase-12, which is transferred to the ER membrane during ER stress in response to the mobilization of intracellular Ca²⁺ stores [20, 22]. Once activated, caspase-12 acts on the effector caspases to induce apoptosis [23]. We also found the highest protein level of caspase-12 at 48 h after curcumin treatment in this study. Furthermore, the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca²⁺ concentration were also found to be affected by curcumin [24].

In order to elucidate the mechanism that how Ca²⁺ was involved in the curcumin-induced cell death, human HCC J5 cells were pretreated with BAPTA, a calcium chelator, followed by the curcumin treatment. The result showed that BAPTA could reverse curcumin-induced cell death, despite the fact that ER stress is able to activate apoptosis [25]. Although the previous study of curcumin-induced apoptosis in HCC J5 cells displays increased ER stress hallmark GADD153 [12], our finding implicates the major significance of Ca²⁺-dependent mechanism in curcumin-induced apoptosis. A similar result has been suggested by Bae et al. in human leukemia cell line as well [7]. Notably, BAPTA also inhibited the depletion of mitochondria membrane potential, ROS production, and capase-3 activation in human HCC J5 cells. In conclusion, our results suggest that the apoptosis induced by curcumin in human HCC J5 cells is through mitochondria-dependent pathway, in which Ca²⁺ release plays an important role.

Conflict of Interests

The authors declare no conflict of interests.

Author’s Contribution

W.-H. Wang and I.-T. Chiang contributed equally to this paper.

Acknowledgment

This paper was supported by Grant NSC99-2623-E-010–001-NU from National Science Council, Taipei, Taiwan.

References

H. Huynh, “Molecularly targeted therapy in hepatocellular carcinoma,” Biochemical Pharmacology, vol. 80, no. 5, pp. 550–560, 2010.
View at: Publisher Site | Google Scholar
R. S. Finn, “Development of molecularly targeted therapies in hepatocellular carcinoma: where do we go now?” Clinical Cancer Research, vol. 16, no. 2, pp. 390–397, 2010.
View at: Publisher Site | Google Scholar
Y. C. Lin, H. W. Chen, Y. C. Kuo, Y. F. Chang, Y. J. Lee, and J. J. Hwang, “Therapeutic efficacy evaluation of curcumin on human oral squamous cell carcinoma xenograft using multimodalities of molecular imaging,” American Journal of Chinese Medicine, vol. 38, no. 2, pp. 343–358, 2010.
View at: Publisher Site | Google Scholar
J. J. Johnson and H. Mukhtar, “Curcumin for chemoprevention of colon cancer,” Cancer Letters, vol. 255, no. 2, pp. 170–181, 2007.
View at: Publisher Site | Google Scholar
C. C. Su, J. S. Yang, C. C. Lu et al., “Curcumin inhibits human lung large cell carcinoma cancer tumour growth in a murine xenograft model,” Phytotherapy Research, vol. 24, no. 2, pp. 189–192, 2010.
View at: Publisher Site | Google Scholar
D. R. Green and J. C. Reed, “Mitochondria and apoptosis,” Science, vol. 281, no. 5381, pp. 1309–1312, 1998.
View at: Google Scholar
J. H. Bae, J. W. Park, and T. K. Kwon, “Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca²⁺ depletion and cytochrome c release,” Biochemical and Biophysical Research Communications, vol. 303, no. 4, pp. 1073–1079, 2003.
View at: Publisher Site | Google Scholar
A. Deniaud, O. Sharaf El Dein, E. Maillier et al., “Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis,” Oncogene, vol. 27, no. 3, pp. 285–299, 2008.
View at: Publisher Site | Google Scholar
J. Cao, Y. Liu, L. Jia et al., “Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells,” Free Radical Biology and Medicine, vol. 43, no. 6, pp. 968–975, 2007.
View at: Publisher Site | Google Scholar
J. Bakhshi, L. Weinstein, K. S. Poksay, B. Nishinaga, D. E. Bredesen, and R. V. Rao, “Coupling endoplasmic reticulum stress to the cell death program in mouse melanoma cells: effect of curcumin,” Apoptosis, vol. 13, no. 7, pp. 904–914, 2008.
View at: Publisher Site | Google Scholar
P.-C. Liao, S.-K. Tan, C.-H. Lieu, and H.-K. Jung, “Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis,” Journal of Cellular Biochemistry, vol. 104, no. 4, pp. 1509–1523, 2008.
View at: Publisher Site | Google Scholar
C. Y. Cheng, Y. H. Lin, and C. C. Su, “Curcumin inhibits the proliferation of human hepatocellular carcinoma J5 cells by inducing endoplasmic reticulum stress and mitochondrial dysfunction,” International Journal of Molecular Medicine, vol. 26, no. 5, pp. 673–678, 2010.
View at: Publisher Site | Google Scholar
S. S. Lin, H. P. Huang, J. S. Yang et al., “DNA damage and endoplasmic reticulum stress mediated curcumin-induced cell cycle arrest and apoptosis in human lung carcinoma A-549 cells through the activation caspases cascade- and mitochondrial-dependent pathway,” Cancer Letters, vol. 272, no. 1, pp. 77–90, 2008.
View at: Publisher Site | Google Scholar
Z. Xia, B. Lundgren, A. Bergstrand, J. W. DePierre, and L. Nässberger, “Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and Bcl-X(L),” Biochemical Pharmacology, vol. 57, no. 10, pp. 1199–1208, 1999.
View at: Publisher Site | Google Scholar
S. W. Ip, S. Y. Wu, C. C. Yu et al., “Induction of apoptotic death by curcumin in human tongue squamous cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum stress and mitochondria-dependent pathways,” Cell Biochemistry and Function, vol. 29, no. 8, pp. 641–650, 2011.
View at: Google Scholar
C.-L. Kuo, S.-Y. Wu, S.-W. Ip et al., “Apoptotic death in curcumin-treated NPC-TW 076 human nasopharyngeal carcinoma cells is mediated through the ROS, mitochondrial depolarization and caspase-3-dependent signaling responses,” International Journal of Oncology, vol. 39, no. 2, pp. 319–328, 2011.
View at: Publisher Site | Google Scholar
S. Prakobwong, S. C. Gupta, J. H. Kim et al., “Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways,” Carcinogenesis, vol. 32, no. 9, pp. 1372–1380, 2011.
View at: Publisher Site | Google Scholar
W. Dröge, “Free radicals in the physiological control of cell function,” Physiological Reviews, vol. 82, no. 1, pp. 47–95, 2002.
View at: Google Scholar
H. R. Rezvani, F. Mazurier, M. Cario-André et al., “Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes,” Journal of Biological Chemistry, vol. 281, no. 26, pp. 17999–18007, 2006.
View at: Publisher Site | Google Scholar
S. Orrenius, B. Zhivotovsky, and P. Nicotera, “Regulation of cell death: the calcium-apoptosis link,” Nature Reviews Molecular Cell Biology, vol. 4, no. 7, pp. 552–565, 2003.
View at: Publisher Site | Google Scholar
K. Xu, N. Tavernarakis, and M. Driscoll, “Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca²⁺ release from the endoplasmic reticulum,” Neuron, vol. 31, no. 6, pp. 957–971, 2001.
View at: Publisher Site | Google Scholar
T. Nakagawa, H. Zhu, N. Morishima et al., “Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β,” Nature, vol. 403, no. 6765, pp. 98–103, 2000.
View at: Publisher Site | Google Scholar
T. Yoneda, K. Imaizumi, K. Oono et al., “Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress,” Journal of Biological Chemistry, vol. 276, no. 17, pp. 13935–13940, 2001.
View at: Google Scholar
M. Wang, Y. Ruan, Q. Chen, S. Li, Q. Wang, and J. Cai, “Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca²⁺ concentration,” European Journal of Pharmacology, vol. 650, no. 1, pp. 41–47, 2011.
View at: Publisher Site | Google Scholar
S. Oyadomari and M. Mori, “Roles of CHOP/GADD153 in endoplasmic reticulum stress,” Cell Death and Differentiation, vol. 11, no. 4, pp. 381–389, 2004.
View at: Publisher Site | Google Scholar

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Copyright © 2012 Wei-Hsun Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Evidence-Based Complementary and Alternative Medicine

Curcumin-Induced Apoptosis in Human Hepatocellular Carcinoma J5 Cells: Critical Role of C a + 2 -Dependent Pathway

Abstract

1. Introduction

2. Materials and Methods

2.1. Cell Culture

2.2. Morphological Study and Cell Viability

2.3. Comet Assay

2.4. DNA Fragmentation

2.5. Caspase-3 Activity Assay

2.6. Detection of Reactive Oxygen Species (ROS)

2.7. Detection of Mitochondrial Membrane Potential (ΔΨm)

2.8. Cell Viability, ROS Production, ΔΨm Levels in J5 cells Pre-Treated with BAPTA

2.9. Determination of Ca2+ Concentration

2.10. Western Blotting

2.11. Statistics

3. Results

3.1. Morphological Study and Cell Viability

3.2. Ca2+ Production, Mitochondria Membrane Potential (ΔΨm), and Production of Reactive Oxygen Species (ROS) Affected by Curcumin in J5 Cells

3.3. The Release of Cytochrome c and Apoptotic-Associated Proteins Affected by Curcumin in J5 Cells

3.4. DNA Damage and Fragmentation Caused by Curcumin in J5 Cells

3.5. Effects of Calcium Chelator BAPTA on Cell Viability, ΔΨm, ROS Production, and Caspase-3 Activity Induced by Curcumin in J5 Cells

4. Discussion

Conflict of Interests

Author’s Contribution

Acknowledgment

References

Copyright

Curcumin-Induced Apoptosis in Human Hepatocellular Carcinoma J5 Cells: Critical Role of -Dependent Pathway

2.7. Detection of Mitochondrial Membrane Potential (ΔΨ_m)

2.8. Cell Viability, ROS Production, ΔΨ_m Levels in J5 cells Pre-Treated with BAPTA

2.9. Determination of Ca²⁺ Concentration

3.2. Ca²⁺ Production, Mitochondria Membrane Potential (ΔΨ_m), and Production of Reactive Oxygen Species (ROS) Affected by Curcumin in J5 Cells

3.5. Effects of Calcium Chelator BAPTA on Cell Viability, ΔΨ_m, ROS Production, and Caspase-3 Activity Induced by Curcumin in J5 Cells