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Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca2+ concentration
European Journal of Pharmacology 650 (2011) 41–47
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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 induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca2+ concentration Mu Wang a, Yuxia Ruan b, Qian Chen a, Shengpu Li a, Qiulan Wang a, Jiye Cai a,⁎ a b
Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou, 510632, PR China Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou, 510632, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2010 Received in revised form 28 August 2010 Accepted 7 September 2010 Available online 29 September 2010 Keywords: Curcumin Apoptosis Atomic force microscope Intracellular free Ca2+ Mitochondrial membrane potential
a b s t r a c t Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca2+ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma is the most common cancer in some parts of the world, with more than 1 million new cases being diagnosed each year. It is potentially curable by surgical resection and chemotherapy. But surgery is the treatment of choice for only the small fraction of patients with localized disease and chemotherapy drugs can damage normal cells, as well as some other side effects (Mor et al., 1998). It is imperative to develop new agents for treatment of this cancer and requires a more novel and integrated approach for prevention, diagnosis and treatment. Recent attention is focused on phytochemicals as anticancer agents. Curcumin (diferuloylmethane) is a kind of lowmolecular-weight and natural polyphenolic compound isolated from the turmeric rhizome (Curcuma longa). It has phenolic groups and conjugated double bonds (Fig. 1). Besides, it has various properties including antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative, wound healing and antitumor properties (Deshpande and Maru, 1995; Sharma et al., 2005; Maheshwari et al., 2006; Ono et al.,
⁎ Corresponding author. Tel./fax: + 86 20 85223569. E-mail address: [email protected] (J. Cai). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.09.049
2004). Preclinical studies have shown that curcumin can inhibit carcinogenesis in various cell lines such as gastric, colon, breast, cervical, hepatic, and ovarian cancer (Aggarwal et al., 2003). Compared with some chemotherapy drugs, curcumin has the advantage to induce cancer cell apoptosis, while with low cytotoxic effects on normal cells (Syng-ai et al., 2004; Kunwar et al., 2008). Thus, it's a potential compound for drug development against cancer (Hatcher et al., 2008). Although there are many studies of anticancer effect about curcumin, little is known about membrane morphology and mechanism induced by it. Plasma membrane plays a very important role on cell physiology. It's a boundary between live cell and external environment and protects cell from harm (Puech et al., 2006). Also it regulates cell functions and transportation of nutrition inside and outside cell (Alarmo et al., 2009; Heidemann and Wirtz, 2004). Changes of membrane structure have a direct influence on cell functions (Voïtchovsky et al., 2006; Sato et al., 2007). In recent years, studies of the cellular, subcellular and molecular mechanical changes on human disease states, including cancer, have emerged as a topic of rapidly expanding scientific interest. A particular focus is to explore the connections among the cell ultrastructure, cellular and cytoskeletal mechanical properties, biological function and human health/disease (Suresh, 2007). In this paper, atomic force microoscopy was used to visualize cell morphology and the membrane ultrastucture.
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images (256 × 256 pixels) were smoothed by onboard software (IP2.1) in order to remove the background noise at low frequency in scanning direction (flatten order: 0–1).
2.5. Cell apoptosis assay Fig. 1. Chemical structure of curcumin.
Recent studies have demonstrated that cell apoptosis is related to mitochondrial membrane potential, especially for the cancer cells (Scarlett et al., 2000; Hu and Kavanagh, 2003). Mitochondrial membrane potential decrease could result in activation of mitochondrial pro-apoptotic factors (Hu and Kavanagh, 2003). In addition, another factor related to cell death is the intracellular free Ca2+ level, which is companied with decrease of mitochondrial membrane potential, release of cytochrome c from mitochondria and the activation of apoptosis proteins casepase (Simon et al., 2000). In this paper, we evaluated the Ca2+ level and mitochondrial membrane potential in curcumin-induced human hepatocellular carcinoma cell line (HepG2). And the apoptosis mechanism was investigated. 2. Materials and methods 2.1. Materials Human hepatocellular carcinoma cell line (HepG2) was donated by Institute of Physiology, Jinan University. RPMI1640 medium and fetal bovine serum were purchased from Giboc Co. Curcumin was bought from Tianjin Yongda Chemical Reagent Development Center. The cell counting kit was purchased from Dojin Laboratory (Kumamoto, Japan). Fluo-3 AM and Rhodamine 123 were the products of Beyotime Institute of Biotechnology, China. Annexin V-FITC and PI apoptosis detection kit was bought from Keygen Biotechnology, China. 2.2. Cell culture Curcumin was dissolved in dimethylsulphoxide. Final curcumin concentrations of 5–40 μM were obtained by dilution in culture media such that the final concentration of dimethylsulphoxide was not N0.1%. Controls containing 0.1% dimethylsulphoxide were included in all experiments. The cells were maintained in RPMI 1640, 10% (v/v) fetal bovine serum at 37 °C in 95% air and 5% CO2. 2.3. Proliferation and viability assays Cell viability was assessed by a cell counting kit. The method was referred to the literature (Yao et al., 2007). After 24 h treatment, 5 mg/ml 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium (WST-8) in phosphate buffered saline (PBS) was added to each well and incubated for 2 h at 37 °C in 95% air and 5% CO2. Then absorbance was measured in a dual-beam microtiter plate reader (TECAN Model Safire2) at 450 nm with a 600 nm reference. Cell viability rate= (ODtreatment/ODcontrol)×100%. Each experiment group repeated at least three times.
Apoptosis was determined by translocation of phosphatidylserine to the cell surface using an Annexin V-FITC and PI apoptosis detection kit (Nanjing KeyGen Biotech. Co. Ltd., China). After 24 h of treatment with or without curcumin, cells were harvested and washed twice in cold PBS, and resuspended in Annexin V-FITC and PI for 30 min in the dark. Cell apoptosis was analyzed by using Cell Quest software on a FACSAria Flow Cytometer (BD Inc., USA). Fluorescence was detected with an excitation wavelength of 480 nm.
2.6. Mitochondrial membrane potential detection Mitochondrial inner membrane is negatively charged for being rich of negatively charged glycoprotein. A large acumination of protons out of the inner membrane caused transmembrane potential. Mitochondrial Membrane Potential was monitored by the fluorescent dye, Rhodamine 123. It is a cell permeable cationic dye that preferentially enters into mitochondria based on highly negative mitochondrial membrane potential (△Ψm). Depolarization of mitochondrial membrane potential (△Ψm) for cell apoptosis results in the loss of Rhodamine123 from the mitochondria and a decrease in intracellular fluorescence intensity. After 24 h treatment with or without curcumin, cells were harvested and washed twice in cold PBS, then resuspended in Rhodamine 123 (2 μM) for 30 min in dark. Fluorescence was measured by flow cytometry with an excitation wavelength of 485 nm.
2.7. Intracellular free Ca2+ detection The level of intracellular free Ca2+ is decided by using a fluorescent dye Fluo-3 AM which can across the cell membrane and be cut into Fluo-3 by intracellular esterase. The Fluo-3 can specifically combine with the Ca2+ and has a strong fluorescence with an excitation wavelength of 488 nm. After exposed to curcumin (0, 5, 10, 20, 40 μM) for 24 h, HepG2 cells were harvested and washed twice with PBS, then resuspended in Fluo-3 AM (5 μM) for 30 min in dark. Detection of intracellular Ca2+ was carried by Flow cytometer at 525 nm excitation wavelength.
2.8. Statistical analysis Data were presented as mean ± standard deviation and analyzed using Student's t test. All experiments performed at least three times independently.
3. Results 3.1. Effect of curcumin on HepG2 cell viability
2.4. Atomic force microscopy visualization Incubating HepG2 cells with curcumin (5–40 μM) for 24 h were fixed for 15 min with 4% paraformaldehyde after monolayer-cultured, then washed twice to triple and air dried in room temperature. After putting the prepared sample on the XY scanning station of atomic force microscopy (Thermomicroscope), the monitor was used to locate scanning area and contact mode was applied for imaging. In our study, we adopted 100 μm scanner and UL20B Si3N4 probe of which the elasticity coefficient was 2.8 N/m. All of the acquired
The cell counting kit was used to assess the cell proliferation and viability of curcumin-treated HepG2 cells. As shown in Fig. 2, with the concentration of curcumin increased, cell viability was decreased after treated with curcumin for 24 h. Particularly from 10 to 20 μM, the cell viability was decreased significantly, almost dropped about 3 times. The 50% inhibiting concentration (IC50) (17.5 ± 3.2 μM) was located in this area. However, cell viability decreased very little between 20– 40 μM. At 40 μM, the viability of HepG2 cells was about 4 times lower than that of the control cells.
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et al., 2000; Hu and Kavanagh, 2003). In order to gain a better understand of the mechanism of curcumin-induced HepG2 cell apoptosis, we used Rhodamine 123 to acquire the mitochondrial membrane potential by examining its fluorescent intensity. As shown in Fig. 5, the fluorescent intensity decreased with the increase of curcumin concentration. There was a concentration dependent decrease of Rhodamine 123 fluorescence after treated with curcumin, compared with the control group. That indicated curcumin was able to induce mitochondrial membrane potential disruption in HepG2 cells. 3.5. Curcumin induced Ca2+ fluctuation
Fig. 2. Effect of curcumin on HepG2 cell viability. Cell viability was determined by a CCK-8 assay as described in the text. Results are expressed as means ± SD of data obtained in three independent experiments.
3.2. Effect of curcumin on HepG2 cell morphology and ultrastructure As shown in Fig. 3A1, the cells in control group revealed a typical long spindle-shaped morphology and the nuclei were plump and ovalized. The cell tails were unrolled and were a little wider than that of the treated group. Between cells, the pseudopodium connected with each other for material exchange and information transfer. The atomic force microscopy ultrastructure showed that cell membrane was made up of many nanoparticles (Fig. 3C1). Previous work has been reported that the visible protruding particles are clusters of membrane proteins (Christian et al., 1998). After treated with 5 μM curcumin, the cell morphology and ultrastructure were similar to the control group (Fig. 3A2–C2). When curcumin concentration increased to 10 μM, the cell morphology was deformed (Fig. 3A3). As increased to 20 μM, the cells were not plump but shrank. The cell tails also shrank and presented threadiness. These indicated that the intercellular communication reduced. The ultrastructure showed the nanoparticles on membrane were bigger than that in control group (Fig. 3A4–C4). It indicated a disorganized topographic structure of the membrane. In Fig. 3A5, the cells shrank further and became round after treated with 40 μM curcumin. The tail turned into much more slender and intracellular interaction was further reduced. Fig. 3C5 showed that the nanoparticles on cell membrane were not of uniform size. 3.3. Effect of curcumin on HepG2 cell apoptosis Cell apoptosis was determined by Annexin V-FITC and PI apoptosis detection kit. In the apoptosis map (Fig. 4B), the Q2 means the late apoptosis and dead cells, the Q3 was presented the live cells and the Q4 was the early apoptosis cells. As shown in Fig. 4A and B, the cell apoptosis of HepG2 was increased slowly by treated with curcumin for 24 h, especially between 10–20 μM. At 40 μM, the apoptosis was 34.7%. It was more than 3 times of that in control group. Also the percentage of late apoptosis and dead cells increased with the increased concentration. From 10 to 20 μM, it increased significantly and reached 60.2%, which was more than 4 times of that at 10 μmol/L. However, between 20–40 μM, it decreased a little (from 60.2% to 55.6%). Even so, the total rate of cell death (Q2 + Q4) increased (90.3%). 3.4. Curcumin induced disruption of mitochondrial membrane potential It is reported that disruption of mitochondrial membrane potential irreversibly leads to cell apoptosis, results in release of cytochrome c and a decrease of adenosine triphosphate (ATP) generation (Scarlett
A sustained increase in intracellular Ca2+ concentrations is recognized to be a factor for cell death and cell injury (Bano and Nicotera, 2007; Eisner et al., 2006). With this in mind, we used Fluo-3 AM to examine the effects of curcumin on intracellular Ca2+ mobilizations in HepG2 cells. In control group, the level of intracellular free Ca2+ was the lowest. With the curcumin increased, the level of intracellular Ca2+ increased steadily (Fig. 6). As the curcumin concentration decreased from 20 to 40 μM, intracellular free Ca2+ fluorescence increased dramatically (from 673 to 1083). That was in accordance with the tendencies of mitochondrial membrane potential and cell apoptosis. The results indicated that the increase of intracellular Ca2+ was related with curcumin-induced HepG2 cell apoptosis. 4. Discussions Tumor cell resistance to apoptosis is an inherent part of the carcinogenic process and is also implicated in resistance to chemotherapeutic drugs (Johnstone et al., 2002). Therefore, phytochemicals such as curcumin is applied to target resistant cells and improves efficacy without toxicity on normal cells (Syng-ai et al., 2004). Previous studies have reported that curcumin can not induce apoptosis in normal cells including primary cultures. For example, Syng-ai et al. (2004) have studied the cytotoxic effects of curcumin on three human tumor cell lines and rat primary hepatocytes. The results indicated that curcumin concentration less than 50 μM had a very low cytotoxity on normal cells. Kunwar et al. (2008) used two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7 to calculate the cellular uptake and cytotoxicity of curcumin. They found both the uptake and cell inhibition were significantly higher in tumor cells (EL4 and MCF7) compared to the normal cells (spleen lymphocytes and NIH3T3). Their results indicated that curcumin had no toxicity on normal cells, which showed no superoxide generation and therefore no cell death. Our results indicated that there was an increase in the cytotoxicity in HepG2 cells with increasing concentration of curcumin treatment. Existed evidence supported curcumin used in cancer prevention through its antiproliferative and anticarcinogenic properties or as an adjunct in overall cancer treatment. Several studies have suggested that curcumin can induce apoptotic cell death in malignant cells (Karunagaran et al., 2005). But the mechanisms responsible for apoptosis induced by curcumin appear to be ill-defined. Most of the studies declared that apoptotic cell death was related to the stability of oncogene protein p53, the release of cytochrome c and the generation of reactive oxygen species (Aggarwal et al., 2003; Tsvetkov et al., 2005). More recently, treatment with curcumin has been reported to induce autophagic cell death in malignant cells (Aoki et al., 2007). In our study, curcumin could inhibit cell growth and induce apoptosis in HepG2 cells. Cell viability and cell apoptosis had a dosedependent relationship with curcumin concentration respectively. With the curcumin concentration increased, cell viability decreased significantly and the apoptosis increased steadily. However, as for 20– 40 μM, the late apoptosis and dead cells (Q2) decreased a little (Fig. 4).
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Fig. 3. AFM images of HepG2 cells before and after curcumin treatment for 24 h. (A)Topography of HepG2 Cells, Scale bar in A1–A5 is 10 μm; (B)Tail image of HepG2 cells, Scale bar in B1–B5 is 5 μm; (C)Cell membrane ultrastructure of HepG2 cells, Scale bar in C1–C5 is 1 μm; (A1,B1 and C1) Control group; (A2,B2 and C2) 5 μM curcumin treated group; (A3,B3 and C3)10 μM curcumin treated group; (A4,B4 and C4) 20 μM curcumin treated group; (A5,B5 and C5) 40 μM curcumin treated group.
It meant that most of the cells were necrosis rather than apoptosis in this concentration range. Syng-ai et al. (2004) found that after curcumin (50 μM) incubated with normal cells such as lymphocytes, hepatocytes, rat skin fibroblasts, Chinese hamster ovary for 24 h, the
cell apoptosis were 1.6%, 1.0%, 2.6%, 2.8%, respectively. Compared to the curcumin treated group, the apoptosis of untreated cell lines were 1.0%, 0.5%, 0.4%, 1.4%. Thus, curcumin (the concentration less than 50 μM) has very low toxicity on normal cells.
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Fig. 4. Effect of curcumin on apoptosis in HepG2 cells after treated for 24 h. (A) Annexin V-FITC and PI staining for apoptosis detected by flow cytometry; (B) The graph shows the apoptosis rate of (A). Q2 represents the late apoptosis and dead cells, and Q4 represents the early apoptosis. Control group means the cells are not treated with curcumin.
Atomic force microscopy can serve as a valuable tool to elucidate ultrastructural changes in cell-surface topography at the nanoscale level. The realization of the full potential of high-resolution atomic force microscopy imaging has revealed some very important biological events such as exocytosis and endocytosis (Samsuri et al., 2010). These changes on the cell-surface topography may potentially provide novel insights into the control of cell shape and interaction with pericellular matrix during activation of chemical and mechanical signaling pathways (Iscru et al., 2008). To address this point, we analyzed the cell membrane morphology and ultrastructure in order to investigate effect of curcumin on HepG2 cell. Through direct atomic force microscopy measurement of HepG2 cells (Fig. 3), the cell in control was in a typical long spindle-shaped morphology at the single cellular. After treated with curcumin, the cell morphology turned into round, cell tails shrank and presented threadiness. These changes implied curcumin could decrease the capacity of HepG2 cell motility and intracellular communication. With atomic force microscopy at subcellular level, we visualized that after treated with curcumin, particles on cell membrane were bigger than that in control group. It has been reported that the visible protruding particles are clusters of membrane proteins (Christian et al., 1998), which means some biological events such as change of ion channels might have occurred. It might be caused by intracellular Ca2+ concentration increase, however we needed further study. In sum, all these significant changes in cell surface architecture were influenced by treatment
with curcumin. This method provides new insights into study of interaction between drugs and cells. In current research, the opening of mitochondrial permeability transit pore was the direct reason of cell apoptosis. Once the mitochondrial permeability transit pores opened, some molecules would spread into mitochondria non-selectively. It could result in mitochondria depolarization and oxidative phosphorylation uncoupling. Meanwhile adenosine triphosphate (ATP) synthesis is far outweighed the decomposition (Halestrap et al., 2004). Other research indicated that the opening of mitochondrial permeability transit pores could lead to the mitochondrial membrane potential decrease, intracellular Ca2+ concentration increase, and finally the apoptosis mechanism was triggered (Gao et al., 2006). Our results support this view. Increase of intracellular free Ca2+ is mediated by controllable intracellular Ca2+ stores such as endoplasmic reticulum. It controls and fine-tunes the Ca2+ signaling. Ca2+ release from intracellular stores is mainly mediated by two subfamilies of intracellular Ca2+release channels, the inositol 1,4,5-trisphosphate receptors (IP3 receptors) and ryanodine receptors (Sammels et al., 2010). They has been widely documented to be a key modulator of Ca2+ release (Laver, 2007; Györke and Terentyev, 2008; McCarron et al., 2008). The inositol 1,4,5-trisphosphate receptors were activated downstream of the formation of inositol 1,4,5-trisphosphate as a consequence of activation of plasma-membrane receptors and that resulted in the alteration of cell-surface topography at the nanoscale level (Fig. 2).
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Fig. 5. Effect of curcumin on mitochondrial membrane potential. After treated with 0–40 μM curcumin for 24 h, cells determined by flow-cytometric analysis stained with Rhodanmine 123 for 30 min. Results are expressed as relative fluorescent intensity.
The Ryanodine receptors were activated downstream of membrane depolarization either by direct coupling to plasma-membrane voltagedependent Ca2+ channels or by Ca2+-induced Ca2+ release subsequent to Ca2+ influx via these voltage-dependent Ca2+ channels (Sammels et al., 2010). The increase of intracellular free Ca2+ mediated by these channels destructed the mitochondrial membrane potential (as shown in Fig. 5), thus resulted in the release of mitochondrial membrane apoptin and cell apoptosis (Szalai et al., 1999; Hajnoczky et al., 2000; Pacher and Hajnoczky, 2001; Rapizzi et al., 2002; Csordas et al., 2002). That induced the cell-surface molecule distribution and biochemical or biomechanical signal changes, which were reflected in the ultrastucture of cell-surface (Fig. 2). Since the Ca2+-dependent process is one of the common features in the cell death, curcumin may be also useful for treatment of some diseases such as cancer.
5. Conclusions Curcumin not only inhibited the growth of HepG2 cells, but also had a high effect on inducing cell apoptosis without any cytotoxic effects on healthy cells (Hatcher et al., 2008). There existed a dosedependent relationship between the drug concentration and cell apoptosis. Cell morphology shown curcumin could damage the cell membrane and suggested that the degree of damage to a tumor cell membrane has a certain positive correlation with drug concentration. Atomic force microscopy detected ultrastructure indicated that the membrane protein granules gathered to mass after curcumin treated. All these changes disturbed the cell homeostasis and affect cell function including intracellular free Ca2+ concentration and mitochondrial membrane potential. As the curcumin concentration
Fig. 6. Effect of curcumin on intracellular free Ca2+ in HepG2 cells. After treated with 0–40 μM curcumin for 24 h, cells determined by flow-cytometric analysis stained with Fluo-3 AM for 30 min. Results are expressed as relative fluorescent intensity.
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increased from 0 μM to 40 μM, intracellular free Ca2+ fluorescence intensity increased by nearly 3 times and the mitochondrial membrane potential decreased by 25%. All these resulted in the release of mitochondrial membrane apoptin and cell apoptosis, and were reflected in the ultrastructure of cell surface. This research provided us detailed insights into new agents' development for treatment of Hepatocellular carcinoma and mechanism study of curcumin induced HepG2 cell apoptosis. Meanwhile, it could contribute to visual diagnosis of early stage apoptosis in tumor cells in response to anti-cancer drugs, as well as in the studies of the interaction between drugs and cells. However, it required further more research to fully understand the specific anti-cancer mechanism of curcumin. Acknowledgment This work is supported by Ministry of Science and Technology of China (No. 2010CB833603), the National Natural Science Foundation of China (No. 60578025), the Key Project of Chinese Ministry of Education (No. 210254), the Fundamental Research Funds for the Central Universities (No. 21609305), and the Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Education Institutes, Jinan University. References Aggarwal, B.B., Kumar, A., Bharti, A.C., 2003. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res. 23, 363–398. Alarmo, E., Pärssinen, J., Ketolainen, J.M., Savinainen, K., Karhu, R., Kallioniemi, A., 2009. BMP7 influences proliferation, migration, and invasion of breast cancer cells. Cancer Lett. 275, 35–43. Aoki, H., Takada, Y., Kondo, S., Sawaya, R., Aggarwal, B.B., Kondo, Y., 2007. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol. Pharmacol. 72, 29–39. Bano, D., Nicotera, P., 2007. Ca2+ signals and neuronal death in brain ischemia. Stroke 38, 674–676. Christian, L.G., Lesniewska, E., Giocondi, M., Flinot, E., Vié, V., Goudonnet, J.P., 1998. Imaging of the surface of living cells by low-force contact-mode atomic force microscopy. Biophys. J. 75, 695–703. Csordas, G., Madesh, M., Antonsson, B., Hajnoczky, G., 2002. tcBid promotes Ca2+ signal propagation to the mitochondria: control of Ca2+ permeation through the outer mitochondrial membrane. EMBO J. 21, 2198–2206. Deshpande, S.S., Maru, G.B., 1995. Effects of curcumin on the formation of benzo [a] pyrene derived DNA adducts in vitro. Cancer Lett. 96, 71–80. Eisner, D.A., Venetucci, L.A., Trafford, A.W., 2006. Life, sudden death, and intracellular calcium. Circ. Res. 99, 223–224. Gao, S.Y., Wang, Q.J., Ji, Y.B., 2006. Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells. World J. Gastroenterol. 12, 3359–3367. Györke, S., Terentyev, D., 2008. Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc. Res. 77, 245–255. Hajnoczky, G., Csordas, G., Madesh, M., Pacher, P., 2000. Control of apoptosis by IP3 and ryanodine receptor driven calcium signals. Cell Calcium 28, 349–363. Halestrap, A.P., Clarke, S.J., Javadov, S.A., 2004. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc. Res. 61, 372–385.
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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 induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca2+ concentration Mu Wang a, Yuxia Ruan b, Qian Chen a, Shengpu Li a, Qiulan Wang a, Jiye Cai a,⁎ a b
Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou, 510632, PR China Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou, 510632, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2010 Received in revised form 28 August 2010 Accepted 7 September 2010 Available online 29 September 2010 Keywords: Curcumin Apoptosis Atomic force microscope Intracellular free Ca2+ Mitochondrial membrane potential
a b s t r a c t Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca2+ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma is the most common cancer in some parts of the world, with more than 1 million new cases being diagnosed each year. It is potentially curable by surgical resection and chemotherapy. But surgery is the treatment of choice for only the small fraction of patients with localized disease and chemotherapy drugs can damage normal cells, as well as some other side effects (Mor et al., 1998). It is imperative to develop new agents for treatment of this cancer and requires a more novel and integrated approach for prevention, diagnosis and treatment. Recent attention is focused on phytochemicals as anticancer agents. Curcumin (diferuloylmethane) is a kind of lowmolecular-weight and natural polyphenolic compound isolated from the turmeric rhizome (Curcuma longa). It has phenolic groups and conjugated double bonds (Fig. 1). Besides, it has various properties including antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative, wound healing and antitumor properties (Deshpande and Maru, 1995; Sharma et al., 2005; Maheshwari et al., 2006; Ono et al.,
⁎ Corresponding author. Tel./fax: + 86 20 85223569. E-mail address: [email protected] (J. Cai). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.09.049
2004). Preclinical studies have shown that curcumin can inhibit carcinogenesis in various cell lines such as gastric, colon, breast, cervical, hepatic, and ovarian cancer (Aggarwal et al., 2003). Compared with some chemotherapy drugs, curcumin has the advantage to induce cancer cell apoptosis, while with low cytotoxic effects on normal cells (Syng-ai et al., 2004; Kunwar et al., 2008). Thus, it's a potential compound for drug development against cancer (Hatcher et al., 2008). Although there are many studies of anticancer effect about curcumin, little is known about membrane morphology and mechanism induced by it. Plasma membrane plays a very important role on cell physiology. It's a boundary between live cell and external environment and protects cell from harm (Puech et al., 2006). Also it regulates cell functions and transportation of nutrition inside and outside cell (Alarmo et al., 2009; Heidemann and Wirtz, 2004). Changes of membrane structure have a direct influence on cell functions (Voïtchovsky et al., 2006; Sato et al., 2007). In recent years, studies of the cellular, subcellular and molecular mechanical changes on human disease states, including cancer, have emerged as a topic of rapidly expanding scientific interest. A particular focus is to explore the connections among the cell ultrastructure, cellular and cytoskeletal mechanical properties, biological function and human health/disease (Suresh, 2007). In this paper, atomic force microoscopy was used to visualize cell morphology and the membrane ultrastucture.
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images (256 × 256 pixels) were smoothed by onboard software (IP2.1) in order to remove the background noise at low frequency in scanning direction (flatten order: 0–1).
2.5. Cell apoptosis assay Fig. 1. Chemical structure of curcumin.
Recent studies have demonstrated that cell apoptosis is related to mitochondrial membrane potential, especially for the cancer cells (Scarlett et al., 2000; Hu and Kavanagh, 2003). Mitochondrial membrane potential decrease could result in activation of mitochondrial pro-apoptotic factors (Hu and Kavanagh, 2003). In addition, another factor related to cell death is the intracellular free Ca2+ level, which is companied with decrease of mitochondrial membrane potential, release of cytochrome c from mitochondria and the activation of apoptosis proteins casepase (Simon et al., 2000). In this paper, we evaluated the Ca2+ level and mitochondrial membrane potential in curcumin-induced human hepatocellular carcinoma cell line (HepG2). And the apoptosis mechanism was investigated. 2. Materials and methods 2.1. Materials Human hepatocellular carcinoma cell line (HepG2) was donated by Institute of Physiology, Jinan University. RPMI1640 medium and fetal bovine serum were purchased from Giboc Co. Curcumin was bought from Tianjin Yongda Chemical Reagent Development Center. The cell counting kit was purchased from Dojin Laboratory (Kumamoto, Japan). Fluo-3 AM and Rhodamine 123 were the products of Beyotime Institute of Biotechnology, China. Annexin V-FITC and PI apoptosis detection kit was bought from Keygen Biotechnology, China. 2.2. Cell culture Curcumin was dissolved in dimethylsulphoxide. Final curcumin concentrations of 5–40 μM were obtained by dilution in culture media such that the final concentration of dimethylsulphoxide was not N0.1%. Controls containing 0.1% dimethylsulphoxide were included in all experiments. The cells were maintained in RPMI 1640, 10% (v/v) fetal bovine serum at 37 °C in 95% air and 5% CO2. 2.3. Proliferation and viability assays Cell viability was assessed by a cell counting kit. The method was referred to the literature (Yao et al., 2007). After 24 h treatment, 5 mg/ml 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium (WST-8) in phosphate buffered saline (PBS) was added to each well and incubated for 2 h at 37 °C in 95% air and 5% CO2. Then absorbance was measured in a dual-beam microtiter plate reader (TECAN Model Safire2) at 450 nm with a 600 nm reference. Cell viability rate= (ODtreatment/ODcontrol)×100%. Each experiment group repeated at least three times.
Apoptosis was determined by translocation of phosphatidylserine to the cell surface using an Annexin V-FITC and PI apoptosis detection kit (Nanjing KeyGen Biotech. Co. Ltd., China). After 24 h of treatment with or without curcumin, cells were harvested and washed twice in cold PBS, and resuspended in Annexin V-FITC and PI for 30 min in the dark. Cell apoptosis was analyzed by using Cell Quest software on a FACSAria Flow Cytometer (BD Inc., USA). Fluorescence was detected with an excitation wavelength of 480 nm.
2.6. Mitochondrial membrane potential detection Mitochondrial inner membrane is negatively charged for being rich of negatively charged glycoprotein. A large acumination of protons out of the inner membrane caused transmembrane potential. Mitochondrial Membrane Potential was monitored by the fluorescent dye, Rhodamine 123. It is a cell permeable cationic dye that preferentially enters into mitochondria based on highly negative mitochondrial membrane potential (△Ψm). Depolarization of mitochondrial membrane potential (△Ψm) for cell apoptosis results in the loss of Rhodamine123 from the mitochondria and a decrease in intracellular fluorescence intensity. After 24 h treatment with or without curcumin, cells were harvested and washed twice in cold PBS, then resuspended in Rhodamine 123 (2 μM) for 30 min in dark. Fluorescence was measured by flow cytometry with an excitation wavelength of 485 nm.
2.7. Intracellular free Ca2+ detection The level of intracellular free Ca2+ is decided by using a fluorescent dye Fluo-3 AM which can across the cell membrane and be cut into Fluo-3 by intracellular esterase. The Fluo-3 can specifically combine with the Ca2+ and has a strong fluorescence with an excitation wavelength of 488 nm. After exposed to curcumin (0, 5, 10, 20, 40 μM) for 24 h, HepG2 cells were harvested and washed twice with PBS, then resuspended in Fluo-3 AM (5 μM) for 30 min in dark. Detection of intracellular Ca2+ was carried by Flow cytometer at 525 nm excitation wavelength.
2.8. Statistical analysis Data were presented as mean ± standard deviation and analyzed using Student's t test. All experiments performed at least three times independently.
3. Results 3.1. Effect of curcumin on HepG2 cell viability
2.4. Atomic force microscopy visualization Incubating HepG2 cells with curcumin (5–40 μM) for 24 h were fixed for 15 min with 4% paraformaldehyde after monolayer-cultured, then washed twice to triple and air dried in room temperature. After putting the prepared sample on the XY scanning station of atomic force microscopy (Thermomicroscope), the monitor was used to locate scanning area and contact mode was applied for imaging. In our study, we adopted 100 μm scanner and UL20B Si3N4 probe of which the elasticity coefficient was 2.8 N/m. All of the acquired
The cell counting kit was used to assess the cell proliferation and viability of curcumin-treated HepG2 cells. As shown in Fig. 2, with the concentration of curcumin increased, cell viability was decreased after treated with curcumin for 24 h. Particularly from 10 to 20 μM, the cell viability was decreased significantly, almost dropped about 3 times. The 50% inhibiting concentration (IC50) (17.5 ± 3.2 μM) was located in this area. However, cell viability decreased very little between 20– 40 μM. At 40 μM, the viability of HepG2 cells was about 4 times lower than that of the control cells.
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et al., 2000; Hu and Kavanagh, 2003). In order to gain a better understand of the mechanism of curcumin-induced HepG2 cell apoptosis, we used Rhodamine 123 to acquire the mitochondrial membrane potential by examining its fluorescent intensity. As shown in Fig. 5, the fluorescent intensity decreased with the increase of curcumin concentration. There was a concentration dependent decrease of Rhodamine 123 fluorescence after treated with curcumin, compared with the control group. That indicated curcumin was able to induce mitochondrial membrane potential disruption in HepG2 cells. 3.5. Curcumin induced Ca2+ fluctuation
Fig. 2. Effect of curcumin on HepG2 cell viability. Cell viability was determined by a CCK-8 assay as described in the text. Results are expressed as means ± SD of data obtained in three independent experiments.
3.2. Effect of curcumin on HepG2 cell morphology and ultrastructure As shown in Fig. 3A1, the cells in control group revealed a typical long spindle-shaped morphology and the nuclei were plump and ovalized. The cell tails were unrolled and were a little wider than that of the treated group. Between cells, the pseudopodium connected with each other for material exchange and information transfer. The atomic force microscopy ultrastructure showed that cell membrane was made up of many nanoparticles (Fig. 3C1). Previous work has been reported that the visible protruding particles are clusters of membrane proteins (Christian et al., 1998). After treated with 5 μM curcumin, the cell morphology and ultrastructure were similar to the control group (Fig. 3A2–C2). When curcumin concentration increased to 10 μM, the cell morphology was deformed (Fig. 3A3). As increased to 20 μM, the cells were not plump but shrank. The cell tails also shrank and presented threadiness. These indicated that the intercellular communication reduced. The ultrastructure showed the nanoparticles on membrane were bigger than that in control group (Fig. 3A4–C4). It indicated a disorganized topographic structure of the membrane. In Fig. 3A5, the cells shrank further and became round after treated with 40 μM curcumin. The tail turned into much more slender and intracellular interaction was further reduced. Fig. 3C5 showed that the nanoparticles on cell membrane were not of uniform size. 3.3. Effect of curcumin on HepG2 cell apoptosis Cell apoptosis was determined by Annexin V-FITC and PI apoptosis detection kit. In the apoptosis map (Fig. 4B), the Q2 means the late apoptosis and dead cells, the Q3 was presented the live cells and the Q4 was the early apoptosis cells. As shown in Fig. 4A and B, the cell apoptosis of HepG2 was increased slowly by treated with curcumin for 24 h, especially between 10–20 μM. At 40 μM, the apoptosis was 34.7%. It was more than 3 times of that in control group. Also the percentage of late apoptosis and dead cells increased with the increased concentration. From 10 to 20 μM, it increased significantly and reached 60.2%, which was more than 4 times of that at 10 μmol/L. However, between 20–40 μM, it decreased a little (from 60.2% to 55.6%). Even so, the total rate of cell death (Q2 + Q4) increased (90.3%). 3.4. Curcumin induced disruption of mitochondrial membrane potential It is reported that disruption of mitochondrial membrane potential irreversibly leads to cell apoptosis, results in release of cytochrome c and a decrease of adenosine triphosphate (ATP) generation (Scarlett
A sustained increase in intracellular Ca2+ concentrations is recognized to be a factor for cell death and cell injury (Bano and Nicotera, 2007; Eisner et al., 2006). With this in mind, we used Fluo-3 AM to examine the effects of curcumin on intracellular Ca2+ mobilizations in HepG2 cells. In control group, the level of intracellular free Ca2+ was the lowest. With the curcumin increased, the level of intracellular Ca2+ increased steadily (Fig. 6). As the curcumin concentration decreased from 20 to 40 μM, intracellular free Ca2+ fluorescence increased dramatically (from 673 to 1083). That was in accordance with the tendencies of mitochondrial membrane potential and cell apoptosis. The results indicated that the increase of intracellular Ca2+ was related with curcumin-induced HepG2 cell apoptosis. 4. Discussions Tumor cell resistance to apoptosis is an inherent part of the carcinogenic process and is also implicated in resistance to chemotherapeutic drugs (Johnstone et al., 2002). Therefore, phytochemicals such as curcumin is applied to target resistant cells and improves efficacy without toxicity on normal cells (Syng-ai et al., 2004). Previous studies have reported that curcumin can not induce apoptosis in normal cells including primary cultures. For example, Syng-ai et al. (2004) have studied the cytotoxic effects of curcumin on three human tumor cell lines and rat primary hepatocytes. The results indicated that curcumin concentration less than 50 μM had a very low cytotoxity on normal cells. Kunwar et al. (2008) used two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7 to calculate the cellular uptake and cytotoxicity of curcumin. They found both the uptake and cell inhibition were significantly higher in tumor cells (EL4 and MCF7) compared to the normal cells (spleen lymphocytes and NIH3T3). Their results indicated that curcumin had no toxicity on normal cells, which showed no superoxide generation and therefore no cell death. Our results indicated that there was an increase in the cytotoxicity in HepG2 cells with increasing concentration of curcumin treatment. Existed evidence supported curcumin used in cancer prevention through its antiproliferative and anticarcinogenic properties or as an adjunct in overall cancer treatment. Several studies have suggested that curcumin can induce apoptotic cell death in malignant cells (Karunagaran et al., 2005). But the mechanisms responsible for apoptosis induced by curcumin appear to be ill-defined. Most of the studies declared that apoptotic cell death was related to the stability of oncogene protein p53, the release of cytochrome c and the generation of reactive oxygen species (Aggarwal et al., 2003; Tsvetkov et al., 2005). More recently, treatment with curcumin has been reported to induce autophagic cell death in malignant cells (Aoki et al., 2007). In our study, curcumin could inhibit cell growth and induce apoptosis in HepG2 cells. Cell viability and cell apoptosis had a dosedependent relationship with curcumin concentration respectively. With the curcumin concentration increased, cell viability decreased significantly and the apoptosis increased steadily. However, as for 20– 40 μM, the late apoptosis and dead cells (Q2) decreased a little (Fig. 4).
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Fig. 3. AFM images of HepG2 cells before and after curcumin treatment for 24 h. (A)Topography of HepG2 Cells, Scale bar in A1–A5 is 10 μm; (B)Tail image of HepG2 cells, Scale bar in B1–B5 is 5 μm; (C)Cell membrane ultrastructure of HepG2 cells, Scale bar in C1–C5 is 1 μm; (A1,B1 and C1) Control group; (A2,B2 and C2) 5 μM curcumin treated group; (A3,B3 and C3)10 μM curcumin treated group; (A4,B4 and C4) 20 μM curcumin treated group; (A5,B5 and C5) 40 μM curcumin treated group.
It meant that most of the cells were necrosis rather than apoptosis in this concentration range. Syng-ai et al. (2004) found that after curcumin (50 μM) incubated with normal cells such as lymphocytes, hepatocytes, rat skin fibroblasts, Chinese hamster ovary for 24 h, the
cell apoptosis were 1.6%, 1.0%, 2.6%, 2.8%, respectively. Compared to the curcumin treated group, the apoptosis of untreated cell lines were 1.0%, 0.5%, 0.4%, 1.4%. Thus, curcumin (the concentration less than 50 μM) has very low toxicity on normal cells.
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Fig. 4. Effect of curcumin on apoptosis in HepG2 cells after treated for 24 h. (A) Annexin V-FITC and PI staining for apoptosis detected by flow cytometry; (B) The graph shows the apoptosis rate of (A). Q2 represents the late apoptosis and dead cells, and Q4 represents the early apoptosis. Control group means the cells are not treated with curcumin.
Atomic force microscopy can serve as a valuable tool to elucidate ultrastructural changes in cell-surface topography at the nanoscale level. The realization of the full potential of high-resolution atomic force microscopy imaging has revealed some very important biological events such as exocytosis and endocytosis (Samsuri et al., 2010). These changes on the cell-surface topography may potentially provide novel insights into the control of cell shape and interaction with pericellular matrix during activation of chemical and mechanical signaling pathways (Iscru et al., 2008). To address this point, we analyzed the cell membrane morphology and ultrastructure in order to investigate effect of curcumin on HepG2 cell. Through direct atomic force microscopy measurement of HepG2 cells (Fig. 3), the cell in control was in a typical long spindle-shaped morphology at the single cellular. After treated with curcumin, the cell morphology turned into round, cell tails shrank and presented threadiness. These changes implied curcumin could decrease the capacity of HepG2 cell motility and intracellular communication. With atomic force microscopy at subcellular level, we visualized that after treated with curcumin, particles on cell membrane were bigger than that in control group. It has been reported that the visible protruding particles are clusters of membrane proteins (Christian et al., 1998), which means some biological events such as change of ion channels might have occurred. It might be caused by intracellular Ca2+ concentration increase, however we needed further study. In sum, all these significant changes in cell surface architecture were influenced by treatment
with curcumin. This method provides new insights into study of interaction between drugs and cells. In current research, the opening of mitochondrial permeability transit pore was the direct reason of cell apoptosis. Once the mitochondrial permeability transit pores opened, some molecules would spread into mitochondria non-selectively. It could result in mitochondria depolarization and oxidative phosphorylation uncoupling. Meanwhile adenosine triphosphate (ATP) synthesis is far outweighed the decomposition (Halestrap et al., 2004). Other research indicated that the opening of mitochondrial permeability transit pores could lead to the mitochondrial membrane potential decrease, intracellular Ca2+ concentration increase, and finally the apoptosis mechanism was triggered (Gao et al., 2006). Our results support this view. Increase of intracellular free Ca2+ is mediated by controllable intracellular Ca2+ stores such as endoplasmic reticulum. It controls and fine-tunes the Ca2+ signaling. Ca2+ release from intracellular stores is mainly mediated by two subfamilies of intracellular Ca2+release channels, the inositol 1,4,5-trisphosphate receptors (IP3 receptors) and ryanodine receptors (Sammels et al., 2010). They has been widely documented to be a key modulator of Ca2+ release (Laver, 2007; Györke and Terentyev, 2008; McCarron et al., 2008). The inositol 1,4,5-trisphosphate receptors were activated downstream of the formation of inositol 1,4,5-trisphosphate as a consequence of activation of plasma-membrane receptors and that resulted in the alteration of cell-surface topography at the nanoscale level (Fig. 2).
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Fig. 5. Effect of curcumin on mitochondrial membrane potential. After treated with 0–40 μM curcumin for 24 h, cells determined by flow-cytometric analysis stained with Rhodanmine 123 for 30 min. Results are expressed as relative fluorescent intensity.
The Ryanodine receptors were activated downstream of membrane depolarization either by direct coupling to plasma-membrane voltagedependent Ca2+ channels or by Ca2+-induced Ca2+ release subsequent to Ca2+ influx via these voltage-dependent Ca2+ channels (Sammels et al., 2010). The increase of intracellular free Ca2+ mediated by these channels destructed the mitochondrial membrane potential (as shown in Fig. 5), thus resulted in the release of mitochondrial membrane apoptin and cell apoptosis (Szalai et al., 1999; Hajnoczky et al., 2000; Pacher and Hajnoczky, 2001; Rapizzi et al., 2002; Csordas et al., 2002). That induced the cell-surface molecule distribution and biochemical or biomechanical signal changes, which were reflected in the ultrastucture of cell-surface (Fig. 2). Since the Ca2+-dependent process is one of the common features in the cell death, curcumin may be also useful for treatment of some diseases such as cancer.
5. Conclusions Curcumin not only inhibited the growth of HepG2 cells, but also had a high effect on inducing cell apoptosis without any cytotoxic effects on healthy cells (Hatcher et al., 2008). There existed a dosedependent relationship between the drug concentration and cell apoptosis. Cell morphology shown curcumin could damage the cell membrane and suggested that the degree of damage to a tumor cell membrane has a certain positive correlation with drug concentration. Atomic force microscopy detected ultrastructure indicated that the membrane protein granules gathered to mass after curcumin treated. All these changes disturbed the cell homeostasis and affect cell function including intracellular free Ca2+ concentration and mitochondrial membrane potential. As the curcumin concentration
Fig. 6. Effect of curcumin on intracellular free Ca2+ in HepG2 cells. After treated with 0–40 μM curcumin for 24 h, cells determined by flow-cytometric analysis stained with Fluo-3 AM for 30 min. Results are expressed as relative fluorescent intensity.
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increased from 0 μM to 40 μM, intracellular free Ca2+ fluorescence intensity increased by nearly 3 times and the mitochondrial membrane potential decreased by 25%. All these resulted in the release of mitochondrial membrane apoptin and cell apoptosis, and were reflected in the ultrastructure of cell surface. This research provided us detailed insights into new agents' development for treatment of Hepatocellular carcinoma and mechanism study of curcumin induced HepG2 cell apoptosis. Meanwhile, it could contribute to visual diagnosis of early stage apoptosis in tumor cells in response to anti-cancer drugs, as well as in the studies of the interaction between drugs and cells. However, it required further more research to fully understand the specific anti-cancer mechanism of curcumin. Acknowledgment This work is supported by Ministry of Science and Technology of China (No. 2010CB833603), the National Natural Science Foundation of China (No. 60578025), the Key Project of Chinese Ministry of Education (No. 210254), the Fundamental Research Funds for the Central Universities (No. 21609305), and the Key Laboratory of Optoelectronic Information and Sensing Technologies of Guangdong Higher Education Institutes, Jinan University. References Aggarwal, B.B., Kumar, A., Bharti, A.C., 2003. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res. 23, 363–398. Alarmo, E., Pärssinen, J., Ketolainen, J.M., Savinainen, K., Karhu, R., Kallioniemi, A., 2009. BMP7 influences proliferation, migration, and invasion of breast cancer cells. Cancer Lett. 275, 35–43. Aoki, H., Takada, Y., Kondo, S., Sawaya, R., Aggarwal, B.B., Kondo, Y., 2007. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol. Pharmacol. 72, 29–39. Bano, D., Nicotera, P., 2007. Ca2+ signals and neuronal death in brain ischemia. Stroke 38, 674–676. Christian, L.G., Lesniewska, E., Giocondi, M., Flinot, E., Vié, V., Goudonnet, J.P., 1998. Imaging of the surface of living cells by low-force contact-mode atomic force microscopy. Biophys. J. 75, 695–703. Csordas, G., Madesh, M., Antonsson, B., Hajnoczky, G., 2002. tcBid promotes Ca2+ signal propagation to the mitochondria: control of Ca2+ permeation through the outer mitochondrial membrane. EMBO J. 21, 2198–2206. Deshpande, S.S., Maru, G.B., 1995. Effects of curcumin on the formation of benzo [a] pyrene derived DNA adducts in vitro. Cancer Lett. 96, 71–80. Eisner, D.A., Venetucci, L.A., Trafford, A.W., 2006. Life, sudden death, and intracellular calcium. Circ. Res. 99, 223–224. Gao, S.Y., Wang, Q.J., Ji, Y.B., 2006. Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells. World J. Gastroenterol. 12, 3359–3367. Györke, S., Terentyev, D., 2008. Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc. Res. 77, 245–255. Hajnoczky, G., Csordas, G., Madesh, M., Pacher, P., 2000. Control of apoptosis by IP3 and ryanodine receptor driven calcium signals. Cell Calcium 28, 349–363. Halestrap, A.P., Clarke, S.J., Javadov, S.A., 2004. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc. Res. 61, 372–385.
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