Tumor Biol. (2014) 35:753–758 DOI 10.1007/s13277-013-1102-7

RESEARCH ARTICLE

Curcumin induces osteosarcoma MG63 cells apoptosis via ROS/Cyto-C/Caspase-3 pathway Zhengqi Chang & Junchao Xing & Xiuchun Yu

Received: 4 July 2013 / Accepted: 8 August 2013 / Published online: 20 August 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract The antitumor effects of curcumin have attracted widespread attention worldwide. One of its major functions is to induce the apoptosis of tumor cells, but the antitumor mechanism is currently unclear. In the present study, we found that cell mortality and curcumin concentration were dose dependent. Curcumin of low concentrations (10 μΜ) could reduce the level of reactive oxygen species (ROS) in tumor cells, while curcumin of high concentrations (80 μΜ) was able to significantly increase the content of ROS. In addition, Western blotting detection suggested that curcumin of high concentrations can induce the release of Cyto-C and the activation of Caspase-3, and that ROS scavenger NAC apparently inhibits apoptosis protein release and activation, consequently slowing the curcumin-induced apoptosis. Taken together, curcumin further activates the mitochondrial apoptotic pathway by inducing cells to generate ROS and ultimately promotes the apoptosis of tumor cells. Keywords Apoptosis . Curcumin induced . ROS/Cyto-C/ Caspase-3

Introduction Curcuma is a commonly used traditional Chinese medicine, whose main effective component is curcumin with many pharmacological functions [1]. In 1995 in India, Menon et al. [2], for the first time, proposed the possibility that curcumin has antitumor effect. Since then, many investigators have done a lot of work on the antitumor effect and mechanism of curcumin, confirming that curcumin can block the cell Z. Chang : J. Xing : X. Yu (*) Department of Orthopedics, General Hospital of Jinan Military Commanding Region, Jinan 250031, Shandong, People’s Republic of China e-mail: [email protected]

proliferation of various tumors, such as human nasopharyngeal carcinoma cell line NCE [3], gastric cancer cell line SGC7901 [4], and hepatocellular carcinoma cell line HepJ5 [5]. In vivo mouse experiments also indicated that curcumin has a significant antitumor effect [6]. The National Cancer Institute of United States has ranked it as the third generation of anticancer and chemo-preventive agent [7]. It is generally accepted that the antitumor mechanism of curcumin may be related to the induction of tumor cell apoptosis, and the induction way is diverse. Odot et al. found that curcumin induces apoptosis through changing the expression of various oncogene proteins and anti-oncogenes [7, 8]. Anto et al. suggested that curcumin promotes cell apoptosis by regulating apoptosis-related gene (p53) expression [9, 10]. Many studies have found that curcumin can induce cell cycle arrest and eventually lead to apoptosis. Holy reported that after curcumin treatment of human breast cancer cell MCF-7 for 24 h, many cells are arrested in M-phase, and most cells eventually leave M-phase after 48 h, forming multiple micronuclei instead of individual daughter nuclei. This curcumin-induced G2/M arrest is considered to be associated with the aberrant assembly of this nucleus [11]. Squires et al. observed that curcumin inhibits proliferation, invasiveness, and progression through S/G2/M phases of the cell cycle in the non-tumorigenic HBL100 and tumorigenic MDA-MB468 human breast cell lines [12]. Zhu et al. found that cells stops at G1 and G2 phase after curcumin treatment of HepG2 cells [13]. Besides, researchers also found that curcumin can alter Ca2+ and cAMP concentration to regulate intracellular signal transduction and ultimately induces apoptosis [14]. In this study, we selected osteosarcoma MG63 cells as a model. Having used curcumin incubate cells of different concentrations, we found cell mortality and curcumin were concentration dependent. Then, we used the reactive oxygen species (ROS) probe dichlorofluorescin (DCF) to detect the content of intracellular ROS after curcumin incubation of different concentrations. The results of both confocal

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microscope and flow cytometry showed low-concentration curcumin decreased the level of intracellular ROS, whereas high-concentration curcumin significantly increased the content of intracellular reactive oxygen. Detected by Western blotting, it was observed that high-concentration curcumin was capable of inducing Cyto-C release and Caspase-3 activation, and that ROS scavenger Nacetyl-L -cysteine (NAC) inhibited the release and activation of apoptosis protein significantly, ultimately hindering curcumin-induced apoptosis.

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Imaging analysis of living cells

Materials and methods

In order to image the activities of a single cell, a confocal laser scanning microscope system was used. Fluorescent emissions from H2DCFDA (DCF) was monitored confocally using a commercial laser scanning microscope (LSM 510 Meta) combination system (Carl Zeiss, Jena, Germany) equipped with a Plan-Neofluar 40×/1.3 numerical aperture oil differential interference contrast objective. Excitation wavelength and detection filter settings for each of the fluorescent indicators were as follows: DCF fluorescence was excited at 488 nm with an Ar–Ion laser, and the emission was recorded through a 500 to 550-nm band pass filter.

Cell culture

Measurement of intracellular ROS

The human osteosarcoma cell line (MG63) was obtained from the Department of Medicine (Jinan University, Guangzhou, Guangdong, People’s Republic of China). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 15 % fetal calf serum, penicillin (100 U/ ml), and streptomycin (100 mg/ml) in 5 % CO2 at 37 °C in a humidified incubator.

Cellular ROS generation was assayed with DCF. MG63 cells were incubated with 10-μM DCF at 37 °C for 30 min prior to the harvest time point. The cells were then washed, exposed to light, re-suspended in fluorescent activated cell sorting (FACS) buffer (PBS 1 % FBS) and analyzed by flow cytometry using a FAC Sort flow cytometer (BD Biosciences, San Diego, CA). Excitation was set at 488 nm, and emission was recorded on an FL1 detector (525 nm). Electronic compensation was used to eliminate spreading into adjacent fluorescence channels. The data were analyzed with CellQuest software (BD Biosciences, San Diego, CA, USA).

Reagents DMEM was purchased from Invitrogen (Carlsbad, CA). NAC and curcumin were purchased from Sigma. The following fluorophore probes were used: MitoTraker Red (100 nM, Invitrogen Life Technologies, Inc.) to label mitochondria. 2′,7′-dichlorodihydrofluorescein diacetate (2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA), 10 μM, Molecular Probes, Inc.) to detect the production of ROS. Cell viability assays Cell viability was assessed with cell counting kit-8 (CCL-8) (Kumamoto, Japan) after treatment. CCK-8 used highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl) -5-(2,4-disulfophenyl) -2Htetrazoliu, monosodium salt] to produce a water-soluble formazan dye upon reduction in the presence of an electron carrier. Being nonradioactive, CCK-8 allows sensitive determination of the number of viable cells in cell proliferation and cytotoxicity assays. The amount of the formazan dye generated by the activity of dehydrogenases in cells was directly proportional to the number of living cells. At the indicated time, CCK-8 was added to cells and incubated for 1.5 h. OD450, the absorbance value, was read with a 96-well plate reader (DG5032, Hua Dong, Nanjing, China). The value is directly proportional to the number of viable cells in a culture medium (Griffioen and Molema, 2000).

Mitochondrial isolation For mitochondria isolation, cells were harvested and then fractionated using Cytosol/Mitochondria Fractionation Kit (Merch, Germany) according to the supplier’s recommendations. The cytosol extraction and mitochondria were then subjected to western blotting analysis of Cyto-C, respectively. The purity of fractions was tested by immunoblotting with antibodies specific for the cytosolic proteins b-actin or the mitochondrial proteins VCDA1/Porin. Antibodies and western blotting analysis The antibodies used for Western blotting include antibodies against caspase-3 (cleaved), Cyto-C (Cell Signaling Technology, Danvers, MA). At indicated times after PDT, cells were harvested and washed twice with ice-cold PBS (pH 7.4), and lysed with an ice-cold lysis buffer (50-mM Tris–HCl, pH 8.0, 150-mM NaCl, 1 % Triton X-100, and 100 μg/ml phenylmethylsulfonyl fluoride) for 30 min on ice. The lysates were centrifuged at 12,000×g for 5 min at 4 °C, and the protein concentration was determined. Equivalent samples (30 μg of protein extract was loaded on each lane) were fractionated by 12–15 % SDS–PAGE. The proteins were then transferred onto a PDVF membrane (Perkin–Elmer) and

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probed with the indicated antibody, followed by IRDye 800 secondary antibody (Rockland Immunochemicals, Gilbertsville, PA). Detection was performed using the odyssey infra-red imaging system (LI-COR, Lincoln, NE). Equal loading was confirmed with primary antibodies against b-actin (whole-cell lysates) or VCDA1/Porin (mitochondrial preparations). Annexin flow cytometry For flow cytometric analysis (FACS analysis), Annexin-VFITC conjugate and binding buffer were used as standard reagents. Flow cytometry was performed on a FACScanto flow cytometer (Becton Dickinson, Mountain View, CA) with excitation at 488 nm. Fluorescent emission of FITC was measured at 515–545 nm and that of DNA-PI complexes at 564–606 nm. Cell debris was excluded from analysis by an appropriate forward light scatter threshold setting. Compensation was used wherever necessary. Statistics analysis All assays were repeated independently for a minimum of three times. Data are represented as the mean ± SEM. Statistical analysis was performed with Student’s paired t test. Differences were considered statistically significant at P < 0.05.

Results Inhibitory effect of curcumin on the proliferation of human osteosarcoma MG63 cells Curcumin of different concentrations (20, 40, 60, 80 μΜ/L) incubated MG63 cells for 48 h, then detect the survival rates of MG63cells by CCK8. Compared with the control group, the cell survival rates in the curcumin treatment group decreased significantly, and declined with the increase of drug concentration, indicating a dose-dependent manner (Fig. 1).

Fig. 1 Cells viability was analyzed 24 h after various concentrations of curcumin (data represent the mean ± SD of four independent experiments)

of 10-μΜ curcumin incubation group was lower, but it was higher in 40- and 80-μΜ curcumin incubation groups, and additionally, 80-μΜ curcumin incubation group were higher than the 40-μΜ incubation group. The results were in line with those observed under confocal microscopy. Curcumin promotes Cyto-C release and Caspase-3 activation of MG63 cells We extracted the total protein from MG63 cells incubated with 40- and 80-μΜ curcumin for 24 h, and analyzed the release of intracellular Cyto-C and activation of Caspase-3 by Western blotting. As shown in Fig. 3a, compared with the control group, there were obvious release of intracellular Cyto-C and activation of Caspase-3 in curcumin incubation group, with an indication of curcumin concentration dependence. However, there was a significant reduce of Cyto-C release and Caspase-3 activation after adding the ROS scavenger NAC to the culture medium of 80-μΜ curcumin incubation (Fig. 3b).

The influence of curcumin on reactive oxygen in MG63 cells

Apoptosis induced by curcumin

Cells were incubated with 10 and 80-μΜ curcumin, respectively, then confocal microscopy was used to observe the production of intracellular ROS. As shown in Fig. 2a, as compared with the control group, ROS in cells decreased in the low-concentration (10 μΜ) curcumin incubation group as the time was prolonged. Conversely, intracellular ROS increased in the high-concentration (80 μΜ) curcumin incubation group with time extended. Furthermore, we detected the content of intracellular ROS in each group (10, 40, 80 μΜ) by flow cytometry. Compared with the control group, the content

There are three main cell apoptosis pathways: apoptosis, necrosis, and autophagy-associated cell apoptosis. To detect the pathway of cell death involved in the curcumin treatment, the percentage of apoptotic and necrotic cells after curcumin treatment was analyzed by FACS (Fig. 4a). The result suggested that apoptosis was a generally major cell death modality in cells responding to curcumin treatment (Fig. 4b). ROS scavenger NAC attenuated the toxicity of curcumin. It indicated that ROS may contribute to the Cyto-C release and cell apoptosis in the curcumin treatment.

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Fig. 2 The difference of intracellular ROS generation induced by various concentrations curcumin. a Realtime detection of ROS generation by a laser scanning confocal microscopy. Bar =10 μm. b FACS analysis of intracellular ROS generation after the identical dose PDT treatment. Untreated or treated cells were harvested and stained with 10-μM DCF for 30 min in the dark before being analyzed by FACS

Discussion Osteosarcoma is one of the most common malignant bone tumors, with an onset age between 15 to 25 years old. For this highly malignant tumor, women outnumber men, the prognosis is poor, and it is characterized by early pulmonary metastasis. The currently main treatment for osteosarcoma is operation, assisted with chemotherapy and other comprehensive treatments. But the drug resistance of tumor cells has been a barrier of clinical treatment, and some patients react painfully to the chemotherapy, so exploring safe and effective anticancer drugs is a hot topic at present. Curcumin is a plant polyphenol extracted from traditional Chinese medicine turmeric. Studies in recent years found that curcumin not only has the effects of antioxidant, anti-inflammatory, reducing blood lipid [15], but also can inhibit the cell proliferation of a variety of tumors (glioma, breast cancer, lung cancer,

esophageal cancer, ovarian cancer), eventually promoting apoptosis [16, 17]. The antitumor mechanism of curcumin is complex [18]. Recently, great efforts have directed to the research on the mechanism of cell apoptosis induced by curcumin. The results showed that in addition to the obvious inhibition of cell proliferation cycle [19], curcumin can also induce cell apoptosis by means of activating the mitochondrial apoptotic pathway. Zheng et al. found that curcumin can upregulate Caspase-3 and NF-kB protein expression [20]. Zhao et al. suggested that curcumin down-regulates the expression of anti-apoptotic protein Bcl-2 Bcl-XL to promote Caspase-3 expression and induces apoptosis of tumor cells [21]. Bush et al. found curcumin can activate Caspase-3 and Caspase-8 to advance cell apoptosis [22]. To date, however, it remains unclear that how curcumin activates the mitochondrial pathway of apoptosis to stimulate mitochondrial Cyto-C release and Caspase-3 activation.

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Fig. 3 Western blotting analysis of proapoptotic proteins after treatment of various concentrations curcumin. a Immunoblot analysis of proapoptotic proteins (cleaved caspase-3 and cytochrome c). b Quantification of cytochrome c release (data represent mean ± SEM, n =3). c The densities of cleaved caspase-3 bands were measured and the ratio was calculated and compared to the untreated cells (data represent mean ± SEM, n =3)

Fig. 4 The rate of apoptotic cell after treatment of various concentrations curcumin. a Cell death 24 h after curcumin treatment. Cells were stained with FITC-conjugated annexin-V and propidium iodide (PI). b

Quantified analysis of apoptotic and necrotic cell percentage by FACS. All data are representative of three independent experiments

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Previous studies suggest curcumin plays a role in antioxidant, anti atherosclerosis, and the liver and kidneys protection [1]. In the current study, we found that curcumin of low concentration can remove intracellular reactive oxygen and curcumin of high concentration can stimulate intracellular ROS (Fig. 2). Moreover, curcumin-induced Cyto-C release and Caspase-3 activation, and ROS scavenger NAC can significantly inhibit Cyto-C release and Caspase-3 activation induced by high-concentration curcumin (Fig. 3), showing that curcumin further induced the release of apoptosis protein by the induction of intracellular ROS. ROS scavenger NAC obviously hindered apoptosis induced by high-concentration curcumin (Fig. 4). The results proved that curcumin can activate the mitochondrial pathway of apoptosis by inducing intracellular ROS. In summary, there are many apoptosis pathways that are important in curcumin-induced apoptosis. Among them, the mitochondrial pathway is one of the most critical ways. Our results demonstrated that high-concentration curcumin activated the mitochondrial pathway of apoptosis through inducing intracellular ROS generation. The current research may facilitate a clearer understanding of the mechanism of curcumin-induced cell apoptosis, and provide the theoretical basis for the antitumor effects of curcumin. Conflicts of interest None

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