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

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 (ΔΨm), 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,N′,N′-tetraacetic acid (BAPTA), an intracellular calcium chelator, was applied. Cell viability was increased, but ΔΨm, 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.


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][4][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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ depletion and the release of cytochrome c in mouse melanoma cells [10]. We hypothesize that curcumin-induced Ca 2+ release will result in mitochondrial Ca 2+ overuptake to affect mitochondria membrane potential stability. To prove this, we choose BAPTA, an intracellular Ca 2+ chelator, as the inhibitor for mitochondrial Ca 2+ 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 Ca 2+ -regulated mitochondria-dependent pathway.

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% CO 2 humidified incubator).

Morphological Study and Cell
Viability. The J5 cells were cultured in 12-well plates at a density of 2 × 10 5 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:  [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.  (ECL System) and exposed by autoradiography.

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).

Ca 2+ Production, Mitochondria Membrane Potential (ΔΨ m ), and Production of Reactive Oxygen Species (ROS)
Affected by Curcumin in J5 Cells. Figure 2(a) showed that Ca 2+ 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)).

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.

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.

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 Ca 2+ 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][16][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 ([Ca 2+ ] i ) is a universal signaling molecule regulating many cellular functions including apoptosis. In addition, Ca 2+ -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 Ca 2+ 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 Ca 2+ 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 Ca 2+ concentration were also found to be affected by curcumin [24]. In order to elucidate the mechanism that how Ca 2+ 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 Ca 2+ -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 Ca 2+ release plays an important role.