1 ONCOLOGY LETTERS 1: , 2010 loe-emodin, an anthraquinone, in vitro inhibits proliferation and induces apoptosis in human colon carcinoma cells KI-YUN LIN 1,2 and YIH-HUEI UEN 1,3,4 1 Department of Medical Research, Chi-Mei Medical Center, 901 Chung-Hua Road, Yung-Kang City, Tainan 710, 2 Department of iotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, 3 Department of Surgery, Chi-Mei Medical Center, 901 Chung-Hua Road; 4 Department of Electrical Engineering, Southern Taiwan University, 1 Nan-Tai Street, Yung-Kang City, Tainan 710, Taiwan, R.O.C. Received November 19, 2009; ccepted February 5, 2010 DOI: /ol_ bstract. The present study aimed to investigate the anticancer effect of aloe-emodin, an anthraquinone compound present in the leaves of loe vera, on two human colon carcinoma cell lines, DLD-1 and WiDr. Colon carcinoma cells were treated with various concentrations of aloe-emodin for different durations. Cell viability was measured by sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4- methoxy-6-nitro)benzene-sulfonic acid hydrate assay. DN fragmentation was analyzed by agarose gel electrophoresis. Nuclear shrinkage was visualized by Hoechst staining. Western blotting was used to indicate the release of apoptosisinducing factor and cytochrome c from mitochondria and the phosphorylation of id. Caspase-3 and casein kinase II activities were measured by the respective assays. Cell viability analyses showed that aloe-emodin induced cell death in a dose- and time-dependent manner. Notably, the WiDr cells were more sensitive to aloe-emodin than the DLD-1 cells. loe-emodin caused the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by activation of caspase-3 leading to DN fragmentation, nuclear shrinkage and apoptosis. In addition, exposure of colon carcinoma cells to aloe-emodin suppressed the casein kinase II activity in a time-dependent manner and was accompanied by a reduced phosphorylation of id, a downstream substrate of casein kinase II and a pro-apoptotic molecule. These findings showed that the inhibition of casein kinase II activity, the release of apoptosis-inducing factor and cytochrome c, and the caspase-3 activation are involved in aloe-emodin-mediated apoptosis in colon carcinoma cells. Correspondence to: Dr Yih-Huei Uen, Division of General Surgery, Department of Surgery, Chi-Mei Medical Center, 901 Chung-Hua Road, Yung-Kang City, Tainan 710, Taiwan, R.O.C. Key words: aloe-emodin, apoptosis, colon carcinoma cells Introduction loe-emodin is a natural active compound present in the leaves of loe vera (1). Some studies have found that aloe-emodin has numerous biological properties including antiviral, antimicrobial and hepatoprotective activities (2). loe-emodin has been reported to exhibit anticancer activity on neuroectodermal tumors, lung squamous cell carcinoma and hepatoma cells (3-5). loe-emodin has also been shown to inhibit S-phase progression in both a transformed glia and a human glioma cell line, sensitize HeLa cells to s 2 O 3 via the generation of reactive oxygen species, and affect the anticancer activity of cisplatin by blocking the activation of extracellular signalregulated kinase (6-8). However, the effect of aloe-emodin on human colon cancer cells has yet to be investigated. poptosis is an actively regulated process of cell death since its intrinsic pathway involves mitochondria (9). Mitochondrial outer membrane permeabilization in response to cell death triggers (e.g., DN damage) is an important early step which is regulated by cl-2 and controls the release of proteins, such as cytochrome c, from the mitochondria to the cytoplasm where they initiate apoptosis, ultimately leading to cell death (10). poptosis-inducing factor, another mitochondrial protein that is released into the cytosol and nucleus, induces chromatin condensation and DN fragmentation (11). Members of the caspase superfamily are produced and activated, a step that hastens the cell death process involving the caspase-dependent apoptotic pathway (10). Casein kinase II is a conserved and ubiquitous protein serine/threonine kinase engaged in various functions including normal and abnormal cell proliferation (12). Casein kinase II is localized in both the cytoplasm and nuclear compartment of healthy cells, but is predominantly present in the nuclear compartment of cancer cells and has been found to be deregulated in all carcinomas studied thus far (13). Evidence links casein kinase II to apoptosis (14). Casein kinase II plays a role in the modulation of caspase susceptibility with id, a pro-apoptotic member of the cl-2 family. It was shown that casein kinase II phosphorylates id close to the caspase-8 cleavage site, thereby preventing cells from undergoing apoptosis (15). Thus, casein kinase II is an important target for
2 542 lin and uen: loe-emodin-induced apoptosis in colon carcinoma cells the treatment of cancer, as the disruption of casein kinase II activity prevents the phosphorylation of id, thereby allowing the cleavage of id by caspases, followed by apoptosis (13,16). Notably, an extensive down-regulation of casein kinase II by antisense casein kinase II in prostate cancer xenografts was found to eventually lead to a complete disappearance of the tumor (13). To evaluate the anticancer effect of aloe-emodin, we employed two distinct human colon carcinoma cell lines, DLD-1 and WiDr. The observation of DN fragmentation and nuclear morphology, and the examination of the release of apoptosis-inducing factor and cytochrome c showed the apoptotic activity of aloe-emodin. The role of casein kinase II in aloe-emodin-induced apoptosis was also investigated. This study reports for the first time that the natural compound aloe-emodin induces apoptosis in human colon carcinoma cells. Materials and methods loe-emodin. loe-emodin [1,8-dihydroxy 3 (hydroxymethyl)- anthraquinone; CS registry no , EU no , purity 95%] was purchased from Sigma-ldrich Co. (St. Louis, MO, US). It was dissolved in dimethylsulfoxide to a concentration of 18.5 mm and stored at -20 C until use. Cell culture and treatments. Human colon carcinoma cell lines, DLD-1 and WiDr, were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in modified Eagle s medium (MEM) (Sigma- ldrich Co.), supplemented with 10% heat-inactivated fetal bovine serum (Moregate iotech, ulimba QLD, ustralia), 1% MEM non-essential amino acid, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate and 250 ng/ml amphotericin (all from Sigma-ldrich Co.). The two cell lines were grown at 37 C in a humidified atmosphere containing 5% CO 2. Prior to treatment, the cells were grown to 80-90% confluency and starved by incubation in basal medium (MEM + 1% MEM non-essential amino acid) for 24 h. Various concentrations of aloe-emodin ( mm in basal medium) and durations (0, 2, 3, 4, 6, 12, 24 and 48 h) were applied. Cell viability assay. Cell viability was assessed using the XTT [sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4- methoxy-6-nitro)benzene-sulfonic acid hydrate] assay kit (Sigma-ldrich Co.), according to the manufacturer's instructions. The assay was conducted three times, independently. Lactate dehydrogenase activity assay. t the end of the treatment, the culture medium was centrifuged at 250 x g for 10 min, and the supernatant was saved for the lactate dehydrogenase activity assay. The lactate dehydrogenase released from the lysed cells was detected using the CytoTox 96 Non-Radioactive Cytotoxicity assay (Promega, Madison, WI, US), according to the manufacturer's instructions. The assay was conducted three times, independently. DN fragmentation assay. Treated cells were centrifuged and lysed in lysis buffer [10 mm Tris-HCl (ph 8.0), 100 mm NaCl, 1% SDS, 1 mm EDT and 2 mg/ml proteinase K] for 1 h at 65 C. Following two successive extractions with phenol/ chloroform, the DN samples were precipitated in ethanol. fter washing with 70% ethanol, the DN samples were resuspended in TE buffer and subjected to 2% agarose gel electrophoresis. Hoechst staining. Hoechst staining was performed as described in a previous study (17). Hoechst positive nuclei were visualized and photographed using an Olympus fluorescence microscope (Olympus, Tokyo, Japan). Isolation of mitochondria and extraction of mitochondrial proteins. Mitochondria were isolated using a cell mitochondrial isolation kit (Sigma-ldrich Co.), according to the manufacturer's instructions with minor modifications. riefly, treated cells were harvested, resuspended in lysis buffer containing a protease inhibitor cocktail (Sigma-ldrich Co.) and incubated on ice for 1 min. Extraction buffer (1X) containing a protease inhibitor cocktail was added, and the solution was centrifuged at 600 x g for 10 min at 4 C. The supernatant (containing mitochondria) was carefully transferred to a fresh tube and centrifuged at 11,000 x g for 10 min at 4 C. Then, the cytosolic fraction was carefully transferred to a new tube. Mitochondrial proteins were extracted by suspending the pellet in CelLytic M Cell Lysis Reagent (Sigma-ldrich Co.) with a protease inhibitor cocktail. The samples were stored at -80 C until use. The protein content of the cytosolic and mitochondrial fractions was determined using a C protein assay kit (Pierce iotechnology, Inc., Rockford, IL, US) with bovine serum albumin as a standard. Total protein preparation. Total proteins were extracted with an M-PER mammalian protein extraction reagent (Pierce iotechnology, Inc.), according to the manufacturer's instructions. The samples were stored at -80 C until use. The protein concentration was measured as described above. Immunoblotting. Denatured protein samples were subjected to 15% SDS-PGE. Proteins were transferred to nitrocellulose membranes. locked blots were incubated for 1 h at room temperature with the primary antibodies monoclonal antiapoptosis-inducing factor (E-1), monoclonal anti-cytochrome c (-8) (Santa Cruz iotechnology, Inc., Santa Cruz, C, US) and polyclonal anti-phospho id-ser61 (ethyl Laboratories, Inc., Montgomery, TX, US). Monoclonal anti-β-actin (mbcam 8226) (bcam Ltd., Cambridge, UK) was used as a control for equal protein loading. lots were further incubated with secondary antibodies conjugated with alkaline phosphatase for 1 h at room temperature and then incubated with Lumi-Phos W Chemiluminescent substrate (Pierce iotechnology, Inc.) and exposed to a Fuji medical X-ray film (Fuji Photo Film Co., Tokyo, Japan). Image processing was performed using the software Fuji Image Gauge. ll experiments were conducted three times, independently. Caspase-3 activity assay. The caspase-3 activity was determined using a caspase-3 assay kit (Sigma-ldrich Co.). The caspase-3 inhibitor N-acetyl-sp-Glu-Val-sp-CHO (c-devd-cho; iomol Research Laboratories, Plymouth, P, US) was co-treated with aloe-emodin at a final concentration of 200 µm
3 ONCOLOGY LETTERS 1: , Figure 1. loe-emodin is cytotoxic to but does not induce necrosis in the DLD-1 and WiDr colon carcinoma cell lines. The cells were incubated with different concentrations of aloe-emodin for 24 and 48 h. () Survival curve of DLD-1 and WiDr cells. The percentage of viable cells was calculated by defining the viability of cells without aloe-emodin treatment as 100%. () Lactate dehydrogenase release from DLD-1 and WiDr cells. Data are the results of three independent experiments and are presented as the mean ± SD. to inhibit caspase-3 activity in a parallel experiment (18). The assay was conducted three times, independently. Casein kinase II activity assay. Casein kinase II activity was measured using a casein kinase assay kit (Sigma-ldrich Co.) combined with the PKLight HTS protein kinase assay kit (Cambrex io Science Rockland, Inc., Rockland, M, US). riefly, after aloe-emodin treatment, casein kinase II substrate α-casein and a reaction buffer containing TP (both from the casein kinase assay kit) were added to the cell lysates and incubated at 37 C for 15 min. The remaining TP was then consumed by luciferase, and light was generated from TP and luciferin (luciferase and luciferin were from the PKLight HTS protein kinase assay kit). ioluminescence was measured using a luminometer (erthold Detection Systems, Oak Ridge, TN, US). The PKLight HTS protein kinase assay kit measures the consumption of TP and is based on the bioluminescent measurement of the remaining TP present in the samples after kinase activity. The bioluminescent signal is inversely proportional to the activity of the kinase, that is, a higher value is indicative of an increased amount of remaining TP and a lower casein kinase II activity in the cells. The assay was conducted three times, independently. Statistical analysis. The unpaired Student's t-test was used to identify means that were significantly different from each other (P<0.05). Results loe-emodin induced cell death in a dose- and timedependent manner. The effect of aloe-emodin on the viability Figure 2. loe-emodin induces apoptosis in DLD-1 and WiDr cells. Cells were treated with 0.37 mm aloe-emodin for 24 h. () DN fragmentation was analyzed by 2% agarose gel electrophoresis. Lane 1, untreated DLD-1 cells; lane 2, untreated WiDr cells; lane 3, treated DLD-1 cells and lane 4, treated WiDr cells. () poptotic cells were detected by Hoechst staining and examined by fluorescence microscopy. The representative images of three independent experiments are shown (magnification, x400). of DLD-1 and WiDr colon carcinoma cells was examined using the XTT viability assay. Exposure of DLD-1 cells to various concentrations of aloe-emodin (0.04, 0.07, 0.15, 0.22, 0.30 and 0.37 mm) for 24 or 48 h resulted in a dose- and time-dependent decrease in cell viability relative to control cell cultures (Fig. 1). The IC 50 value for 48 h of exposure to aloe-emodin was mm. similar effect of aloeemodin was observed after 24 or 48 h of incubation using the WiDr cell line (Fig. 1). In the latter case, however, the IC 50 value for a 48-h exposure to aloe-emodin was mm, suggesting that the WiDr cells were more sensitive to aloeemodin than the DLD-1 cells. loe-emodin-induced cell death was significant at a concentration of 0.37 mm. Thus, this concentration was applied for the rest of the experiments. loe-emodin did not induce necrosis in DLD-1 and WiDr cells. Treated cells were evaluated for the presence of necrotic cell death by measuring the lactate dehydrogenase release into the medium. Exposure of DLD-1 and WiDr cells to 0.37 mm aloe-emodin for 48 h did not result in the release of lactate dehydrogenase (Fig. 1). loe-emodin induced apoptosis in DLD-1 and WiDr cells. To investigate whether the aloe-emodin-mediated cell death in DLD-1 and WiDr cells was due to an apoptotic mechanism, DN fragmentation and nuclear morphological changes that occurred during aloe-emodin treatment were observed.
4 544 lin and uen: loe-emodin-induced apoptosis in colon carcinoma cells Figure 3. loe-emodin induces the release of apoptosis-inducing factor and cytochrome c (cyt c) in DLD-1 and WiDr cells. Cells were incubated with or without 0.37 mm aloe-emodin for 0, 3, 6, 12 and 24 h. Mitochondrial (mito) and cytosolic (cyto) fractions were analyzed by 15% SDS-PGE and probed with primary antibodies as described in Materials and methods. Results are representative of three independent experiments. Figure 4. loe-emodin activates caspase-3 in DLD-1 and WiDr cells. () Cells were incubated with or without 0.37 mm aloe-emodin for 12 and 24 h. () WiDr cells were incubated with or without 0.37 mm aloe-emodin for 0, 2, 4, 6, 12 and 24 h. To inhibit caspase-3 activity, a caspase-3 inhibitor, c-devd-cho, co-treated with aloe-emodin, was added to WiDr cells at a final concentration of 200 µm. Each bar indicates the value of caspase-3 activity by measuring the release of p-nitroaniline (OD405). Data are the results of three independent experiments and are presented as the mean ± SD. Means are significantly different from the respective control values; * P<0.05 and ** P<0.01. Treatment of DLD-1 and WiDr cells with 0.37 mm aloeemodin for 24 h resulted in DN fragmentation (Fig. 2). Treatment of DLD-1 and WiDr cells with 0.37 mm aloeemodin for 24 h also resulted in changes in the nuclear morphology as evidenced by the Hoechst staining (Fig. 2), a dye often used to label DN in living cells and to observe morphological and nuclear changes (17,19,20). More fragmented nuclei were observed upon aloe-emodin treatment, both in the DLD-1 and WiDr cells. These observations indicated that aloe-emodin-induced cell death in DLD-1 and WiDr cells involved a typical apoptotic pathway. loe-emodin induced the release of apoptosis-inducing factor and cytochrome c. The release of apoptosis-inducing factor and cytochrome c in DLD-1 and WiDr cells upon treatment with aloe-emodin was characterized because these processes are typically related to apoptosis (14). n immunoblot analysis of the mitochondrial and cytosolic fractions derived from aloe-emodin-treated DLD-1 and WiDr cells showed a significant decrease in the amount of apoptosis-inducing factor and cytochrome c in mitochondrial fractions. n increase in apoptosis-inducing factor and cytochrome c in the cytosolic fraction for the indicated time intervals was also noted (Fig. 3). Following treatment of the DLD-1 cells, the release of apoptosis-inducing factor and cytochrome c commenced at 24 h, while that of WiDr cells started after 3 h of treatment. The results were consistent with the cell viability data and confirm that WiDr cells are more sensitive to aloe-emodin than DLD-1 cells. Effect of aloe-emodin on the activation of caspase-3. The involvement of caspase-3 in aloe-emodin-induced apoptosis in DLD-1 and WiDr cells was evaluated. The activity of caspase-3 was significantly induced 12 h after aloe-emodin treatment in WiDr cells (Fig. 4). This effect was also observed in DLD-1 cells albeit only significantly after 24 h of aloe-emodin treatment (Fig. 4). To illustrate the sequence of caspase-3 activation and the release of apoptosis-inducing factor and cytochrome c, a time course of caspase-3 activation after aloe-emodin treatment was performed in WiDr cells. s shown in Fig. 4, caspase-3 activity was significantly induced after 4 h of aloe-emodin treatment. The results showed that the
5 ONCOLOGY LETTERS 1: , (Fig. 5). These results suggest that aloe-emodin induces apoptosis in DLD-1 and WiDr cells through inhibition of the phosphorylation of id. Discussion Figure 5. loe-emodin inhibits the activity of casein kinase II and the phosphorylation of id in DLD-1 and WiDr cells. Cells were incubated with 0.37 mm aloe-emodin for 0, 3, 6 and 12 h. () The activity of casein kinase II was measured as described in Materials and methods. Data are the results of three independent experiments presented as the mean ± SD. Means that are significantly different from the respective control values: ** P<0.01. () Cell lysates were analyzed by 15% SDS-PGE and probed with primary antibodies as described in Materials and methods. Results are representative of three independent experiments. release of apoptosis-inducing factor and cytochrome c started before caspase-3 activation. To demonstrate whether the activation of caspase-3 is essential for aloe-emodin-mediated apoptosis, the influence of the peptidyl inhibitor of caspase-3, c-devd-cho, a generally used caspase-3 inhibitor, was analyzed. loe-emodin-induced caspase-3 activation in WiDr cells was blocked when co-treated with c-devd-cho (Fig. 4). These data showed that the activation of caspase-3 was an essential step in aloe-emodin-induced apoptosis in the two cell lines. However, the DLD-1 cells appeared to be less sensitive than the WiDr cells (data not shown). Effect of aloe-emodin on casein kinase II activity and Ser61 phosphorylation of id. To investigate the role of casein kinase II in aloe-emodin-induced apoptosis, the activity of casein kinase II was examined, as well as the Ser61 phosphorylation of id, a downstream substrate of casein kinase II (Ser61 is one of the phosphorylation sites of id). The activity of casein kinase II started to decrease at 3 h of exposure to aloe-emodin and continued to decrease in a time-dependent manner in WiDr cells (Fig. 5). similar effect was observed in the DLD-1 cells (Fig. 5); however, the activity of casein kinase II started to decrease after 12 h of exposure to aloeemodin. Using anti-phospho id-ser61 polyclonal antibodies, immunoblotting was used to confirm the kinase activity. The aloe-emodin-mediated inhibition of casein kinase II activity caused an inhibition of the Ser61 phosphorylation of id The present study demonstrated that aloe-emodin exhibits anticancer activity in DLD-1 and WiDr colon carcinoma cells and that the cytotoxic mechanism involves the induction of apoptosis. The latter was characterized by the release of apoptosis-inducing factor and cytochrome c from the mitochondria to the cytosol, activation of caspase 3 and inhibition of the activity of casein kinase II, with the subsequent inhibition of the downstream phosphorylation of id. It was reported that, using the human lung squamous carcinoma cell line CH27, apoptosis induced by aloe-emodin was associated with changes in the expression of members of the cl-2 family apoptosis regulators (i.e., increase in the ax level and decrease in cl-xl). loe-emodin was found to cause cytochrome c release from mitochondria (21). In addition, it was observed that aloe-emodin-induced apoptosis in human hepatocellular carcinoma cell lines HepG2 and Hep3 was accompanied by the induction of p53 and p21 expression (5). nother report suggests that the decrease in the expression of protein kinase Cδ and ε isoforms plays a critical role in aloe-emodin-induced apoptosis (21). similar role of protein kinase C was observed in U-937 glioma cells (6). With the exception of protein kinase Cα, most of the protein kinase C isozymes (protein kinase Cα, βi, γ, δ, ε, θ, ξ and μ) were found to decrease upon aloe-emodin treatment. Notably, two hydrophobic residues unique to casein kinase II, Val66 and Ile174, were found to be essential for the interaction with aloe-emodin; a Val66la casein kinase II variant exhibited a substantially affected interaction with aloe-emodin (22,23). In the present study, aloe-emodin-induced apoptosis in colon carcinoma cells involved inhibition of casein kinase II activity and blockage of the phosphorylation of id. We also demonstrated that aloe-emodin resulted in the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by the activation of caspase-3. It is known that phosphorylated id is cleaved by caspase-8, and truncated id binds to mitochondria to induce the release of apoptosis-inducing factor and cytochrome c from mitochondria (10,24,25). It is also known that, in the cytosol, cytochrome c mediates the allosteric activation of pf-1, which is required for the proteolytic maturation of caspase-9 and -3 (10,26). Thus, the inhibition of casein kinase II activity following aloe-emodin treatment may lead to the activation of caspase-3 through the phosphorylation of id and release of apoptosis-inducing factor and cytochrome c from mitochondria. similar pathway was described for Radix Paeoniae lba extract-induced apoptosis in HL-60 leukemic cells and for quercitin-mediated cell apoptosis in murine melanoma 16-L6 cells (27,28). hmad et al found that the down-regulation of casein kinase II by antisense RN elicited intracellular hydrogen peroxide production, which was associated with caspase-3 activation (29). lthough not relative to aloe-emodin, this is another possible pathway between casein kinase II and caspase-3. Thus, further studies are required to elucidate the relationship between casein kinase II and caspase-3 activities.
6 546 lin and uen: loe-emodin-induced apoptosis in colon carcinoma cells Notably, in all of the assays used in the present study, the WiDr colon carcinoma cells were significantly more sensitive to aloe-emodin than the DLD-1 cells. The reason for this difference in sensitivity remains unclear. Controversial data have been analyzed to attempt to correlate differences in response to a given drug with the differentiation state of a given cell line. In an in vitro system using epidermal cells, maturing basal cells were found to undergo apoptosis upon drug treatment, whereas stem cells appeared to be resistant (30). In addition, it has been suggested that undifferentiated keratinocyte stem cells are protected from spontaneous apoptosis (31). Similarly, differentiated human neural crest-derived tumor cells exhibit an increased resistance to potassium ionophore-mediated apoptosis. Cells and human keratinocytes that are undifferentiated are more sensitive to H 2 O 2 -induced cell death than their differentiated counterparts (32,33). However, it was shown that the TNF-related apoptosis-inducing ligand triggers apoptosis in undifferentiated but not in differentiated human keratinocytes (34). Microscopy analyses have demonstrated that DLD-1 cells are less differentiated than WiDr cells (data not shown). Thus, our results strongly suggest that the degree of differentiation of colon carcinoma cells affects the cellular response to aloe-emodin. In addition to the degree of differentiation of culture cells, evidence shows that p53-mutant cell lines are less sensitive to aloe-emodin than their p53 wild-type counterparts (35). In the present study, the amount of mutant p53 in the DLD-1 cells was seven times higher than that in the WiDr cells (1.96 and 0.27 ng/ml cell lysate, respectively). Our data are consistent with the abovementioned study and suggest that the p53 phenotype affects the cellular response to aloe-emodin. In conclusion, using two human colon carcinoma cell lines, we investigated the anticancer effect of the natural product, aloe-emodin, isolated from loe vera leaves. We demonstrated for the first time that aloe-emodin exhibits an anticancer effect against colon carcinoma. cknowledgements The authors would like to thank Dr Sheng-Hsuan Chen, Dr Chun-Chao Chang and Dr Chia-Lang Fang for the helpful comments and review of the manuscript. References 1. Reynolds T: The compounds in loe leaf exudates: a review. ot J Linn Soc 90: , Eshun K and He Q: loe vera: a valuable ingredient for the food, pharmaceutical and cosmetic industries a review. 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Aloe-emodin Induces Cell Death through S-Phase Arrest and Caspase-dependent Pathways in Human Tongue Squamous Cancer SCC-4 Cells TSAN-HUNG CHIU 1*, WAN-WEN LAI 2*, TE-CHUN HSIA 3, JAI-SING YANG 4, TUNG-YUAN
Apoptosis Mediated Cytotoxicity of Curcumin Analogues PGV-0 and PGV-1 in WiDr Cell Line Endah Puji Septisetyani, Muthi Ikawati, Barinta Widaryanti and Edy Meiyanto* ) Cancer Chemoprevention Research Center,
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
ab65320 Mitochondria/Cytosol Fractionation Kit Instructions for Use For the rapid, sensitive and accurate isolation of Mitochondrial and Cytosolic fractions from living cells. This product is for research
ab110168 Mitochondria Isolation Kit for Tissue Instructions for Use For mitochondria isolations from mammalian tissue samples This product is for research use only and is not intended for diagnostic use.
Supplementary Information POLO-LIKE KINASE 1 FACILITATES LOSS OF PTEN-INDUCED PROSTATE CANCER FORMATION X. Shawn Liu 1, 3, Bing Song 2, 3, Bennett D. Elzey 3, 4, Timothy L. Ratliff 3, 4, Stephen F. Konieczny
Mitochondrial DNA Isolation Kit Catalog Number KA0895 50 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials
TFEB-mediated increase in peripheral lysosomes regulates Store Operated Calcium Entry Luigi Sbano, Massimo Bonora, Saverio Marchi, Federica Baldassari, Diego L. Medina, Andrea Ballabio, Carlotta Giorgi
ISSN: 2319-7706 Volume 3 Number 10 (2014) pp. 169-176 http://www.ijcmas.com Original Research Article In-vitro assay for Cytotoxicity activity in ethonolic extract of fruit rind of Couropita Guianensis
SUPPLEMENTARY INFORMATION doi:1.138/nature9814 a A SHARPIN FL B SHARPIN ΔNZF C SHARPIN T38L, F39V b His-SHARPIN FL -1xUb -2xUb -4xUb α-his c Linear 4xUb -SHARPIN FL -SHARPIN TF_LV -SHARPINΔNZF -SHARPIN
#19 Apoptosis Chapter 9 Neelu Yadav PhD Neelu.Yadav@Roswellpark.org Why cells decide to die? - Stress, harmful, not needed - Completed its life span Death stimulation or Stress Cell Survival Death Functions
Silva et al. PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability Supplementary Table; Supplementary Figures and legends S1-S21; Supplementary
3URGXFW,QIRUPDWLRQ Sigma TACS Annexin V Apoptosis Detection Kits Instructions for Use APO-AB Annexin V-Biotin Apoptosis Detection Kit 100 tests For Research Use Only. Not for use in diagnostic procedures.
Anti-Apoptotic Effects of Cellular Therapy Jason Lapetoda 1, Lee K Landeen, PhD 1, George K Michalopoulos, MD 2, Patricia W Bedard, PhD 1 1 Vital Therapies, Inc., San Diego, CA, USA 2 University of Pittsburgh
xcelligence System Real-Time Cell Analyzer Focus Application Compound-Induced Cytotoxicity For life science research only. Not for use in diagnostic procedures. Featured Study: Using the Time Resolving
TALAB Symposium 2007. 11. 9. National Taiwan Univ. Cancer preventive effects of lactic acid bacteria Hyong Joo Lee Seoul National University 1 Total deaths from cancer and heart disease J. Natl. Cancer
TECHNICAL MANUAL Glucose Uptake-Glo Assay Instructions for Use of Products J1341, J1342, and J1343 Revised 2/17 TM467 Glucose Uptake-Glo Assay All technical literature is available at: www.promega.com/protocols/
Purification and Fluorescent Labeling of Exosomes Asuka Nanbo 1*, Eri Kawanishi 2, Ryuji Yoshida 2 and Hironori Yoshiyama 3 1 Graduate School of Medicine, Hokkaido University, Sapporo, Japan; 2 Graduate
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
The Annexin V Apoptosis Assay Development of the Annexin V Apoptosis Assay: 1990 Andree at al. found that a protein, Vascular Anticoagulant α, bound to phospholipid bilayers in a calcium dependent manner.
Supplementary Materials and Methods Reagents and antibodies was purchased from iaffin GmbH & Co KG. Cisplatin (ristol-myers Squibb Co.) and etoposide (Sandoz Pharma Ltd.) were used. Antibodies recognizing
Measurement of PDH Endogenous Activity Relative to the Fully- and Dephosphorylated Phosphorylated States Table of contents PDH Protocol #1 Measurement of PDH Endogenous Activity 1. Introduction 3 2. Regulation
Supplementary Figure S1. Venn diagram analysis of mrna microarray data and mirna target analysis. (a) Western blot analysis of T lymphoblasts (CLS) and their exosomes (EXO) in resting (REST) and activated
Detection of Apoptosis in Primary Cells by Annexin V Binding Using the Agilent 2100 Bioanalyzer Application Note Samuel D. H. Chan Marc Valer and Tobias Preckel, Introduction The Agilent 2100 bioanalyzer
Electrical Stimulation Control Nerve Regeneration via the p38 Mitogen-activated Protein Kinase and CREB Kenji Kawamura, Yoshio Kano. Kibi International University, Takahashi-city, Japan. Disclosures: K.
Biomedical Research 2016; 27 (1): 181-185 ISSN 0970-938X www.biomedres.info Bakuchiol inhibits cell proliferation and induces apoptosis and cell cycle arrest in SGC-7901 human gastric cancer cells. Jing
Chen Accepted: et al.: February Berberine 24, Sensitizes 2015 Ovarian Cancer Cells to Cisplatin www.karger.com/cpb 956 1421-9778/15/0363-0956$39.50/0 Original Paper This is an Open Access article licensed
PUMA gene transfection can enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells C.-G. Sun 1 *, J. Zhuang 1 *, W.-J. Teng 1, Z. Wang 2 and S.-S. Du 3 1 Department of Oncology,
Collagenase Assay Kit Catalog # 31 and 32 For Research Use Only - Not Human or Therapeutic Use INTRODUCTION Collagenases are members of the matrix metalloproteinase (MMP) family and degrade collagen types
MPO Peroxidation Assay Kit Catalog Number KA1338 96 assays Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 Principle of the Assay... 3 General
Supplementary Fig. S1. Evaluation of the purity and maturation of macrophage cultures tested by flow cytometry. The lymphocytic/monocytic cellular fraction was isolated from buffy coats of healthy donors
Clinical significance of CD44 expression in children with hepatoblastoma H.-Y. Cai 1 *, B. Yu 1 *, Z.-C. Feng 2, X. Qi 1 and X.-J. Wei 1 1 Department of General Surgery, General Hospital of Beijing Military
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ab139485 CytoPainter Golgi/ER Staining Kit Instructions for Use Designed to detect Golgi bodies and endoplasmic reticulum by microscopy This product is for research use only and is not intended for diagnostic
Nuclear Extraction Kit Item No. 10009277 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION 3 Materials
BIOCHEMICAL EXAMINATION OF ACUTE MYOCARDIAL INFARCTION 1 Structure of heart muscle Written by Lenka Fialová, translated by Jan Pláteník Heart muscle (myocardium) is a particular form of striated muscle,
METABOLISM Transform Your Metabolic Research COMPREHENSIVE TOOLS FOR ADVANCING YOUR METABOLIC RESEARCH Metabolism refers to a set of chemical reactions which are responsible for transforming carbohydrates,
This document is available at www.stemcell.com/pis Catalog #18765 EasySep Mouse CD4+CD62L+ T Cell Isolation Kit For processing 1x 10^9 cells Description Isolate highly purified naïve CD4+ T cells (CD4+CD62L+)
E.Z.N.A. SQ Blood DNA Kit II Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Blood Storage and DNA Yield...4 Preparing Reagents...5 100-500 μl Whole Blood Protocol...6
EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric) Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Total Histone H3 Acetylation Detection Fast Kit (Colorimetric)
This document is available at www.stemcell.com/pis Catalog #18765 EasySep Mouse CD4+CD62L+ T Cell Isolation Kit For processing 1x 10^9 cells Description Isolate highly purified naïve CD4+ T cells (CD4+CD62L+)
Biol403 MAP kinase signalling The mitogen activated protein kinase (MAPK) pathway is a signalling cascade activated by a diverse range of effectors. The cascade regulates many cellular activities including
Supplementary figure legends Supplementary Figure 1. Exposure of CRT occurs independently from the apoptosisassociated loss of the mitochondrial membrane potential (MMP). (A) HeLa cells treated with MTX
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Molecules 215, 2, 346-343; doi:1.339/molecules22346 Article OPEN ACCESS molecules ISSN 142-349 www.mdpi.com/journal/molecules Anti-Proliferative Effect and Induction of Apoptosis in Androgen-Independent
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Molecules 2002, 7, 896-901 molecules ISSN 1420-3049 http://www.mdpi.org/ Determination of Aldehyde Dehydrogenase (ALDH) Isozymes in Human Cancer Samples - Comparison of Kinetic and Immunochemical Assays
ab178779 Mitochondrial Yeast Isolation Kit Instructions for Use For the isolation of mitochondria from P. pastoris and other species of yeast This product is for research use only and is not intended for
Supplemental Information Tobacco-specific Carcinogen Induces DNA Methyltransferases 1 Accumulation through AKT/GSK3β/βTrCP/hnRNP-U in Mice and Lung Cancer patients Ruo-Kai Lin, 1 Yi-Shuan Hsieh, 2 Pinpin
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Research Foundation, 18 month progress report THE ROLE OF ALTERED CALCIUM AND mtor SIGNALING IN THE PATHOGENESIS OF CYSTINOSIS Ekaterina Ivanova, doctoral student Elena Levtchenko, MD, PhD, PI Antonella
TMM,5-2011 PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS Ice-cold means cooled in ice water. In order to prevent proteolysis, make sure to perform all steps on ice. Pre-cool glass homogenizers, buffers
(2003) 10, 516 527 & 2003 Nature Publishing Group All rights reserved 1350-9047/03 $25.00 www.nature.com/cdd TGF-b1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production
Apoptotic Pathways in Mammals Douglas R. Green 1 Apoptosis A form of cell death that is defined morphologically, and features a number of biochemical events Programmed cell death Cell death that occurs
ab139469 E3 Ligase Auto- Ubiquitilylation Assay Kit Instructions for Use For testing ubiquitin E3 ligase activity through assessment of their ability to undergo auto-ubiquitinylation This product is for
AATEX 14, Special Issue, 699-703 Proc. 6th World Congress on Alternatives & Animal Use in the Life Sciences August 21-25, 2007, Tokyo, Japan HL-60 ATP assay for predicting rat oral toxicity study Yumiko
210 A novel bfgf antagonist peptide inhibits breast cancer cell growth QUCHOU LI 1, SUSU GAO 1, YONGLIN YU 1, WENHUI WANG 1, XILEI CHEN 1, RUIXUE WANG 1, TAO LI 1, CONG WANG 1, XIAOKUN LI 1,2 and XIAOPING
Supplementary Information Supplementary Fig. 1. Elevated Usp9x in melanoma and NRAS mutant melanoma cells are dependent on NRAS for 3D growth. a. Immunoblot for Usp9x protein in NRAS mutant melanoma cells
Depleting Lipoproteins from Serum Kathy K. Foxx Kalen Biomedical LLC President For decades, fetal bovine serum (FBS) has been used as a supplement for cell-culture media, providing the growth factors that
INSTRUCTION MANUAL Quick-cfDNA Serum & Plasma Kit Catalog No. D4076 Highlights High-quality DNA, including cell-free, is easily and robustly purified from up to 10 ml of serum/plasma, up to 1 ml amniotic
Global Histone H3 Acetylation Assay Kit Catalog Number KA0633 96 assays Version: 06 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle
Determination of the temporal pattern and importance of BALF1 expression in Epstein-Barr viral infection Melissa Mihelidakis May 6, 2004 7.340 Research Proposal Introduction Apoptosis, or programmed cell
ab133127 LDL Uptake Assay Kit (Cell-Based) Instructions for Use For the detection of LDL uptake into cultured cells. This product is for research use only and is not intended for diagnostic use. Version
Wang et al. BMC Cancer 2014, 14:37 RESEARCH ARTICLE Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxelresistant MCF-7 breast cancer cells Harris Wang 1, The Vo 1, Ali Hajar
MANUAL IL-1alpha (mouse) ELISA Kit [Interleukin-1 alpha (mouse) ELISA Kit] For research use only. Not for diagnostic use Version 1 (March-5-2013) Cat. No. AG-45B-0003-KI01 www.adipogen.com Table of Contents
Table S1. Sequence of human and mouse primers used for RT-qPCR measurements. Ca9, carbonic anhydrase IX; Ndrg1, N-myc downstream regulated gene 1; L28, ribosomal protein L28; Hif1a, hypoxia inducible factor
- 1 - CITROZINE Investigations on its antioxidative and CITROFRESH SUPERCONCENTRATE anti-inflammatory potential Investigator and responsible for the correctness of the test protocol, results, conclusions
Chapter 4 Estrogen receptor expression in human macrophages 4.1. Introduction Macrophages respond to estrogen present in their microenvironment and hence should express functional estrogen receptors unless
Supplemental material Edens and Levy, http://www.jcb.org/cgi/content/full/jcb.201406004/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Nuclear shrinking does not depend on the cytoskeleton
INCUCYTE LIVE-CELL ANALYSIS SYSTEM Assays for Immuno-oncology Research Real-time automated measurements of immune and tumor cell dynamics within your incubator See what your cells are doing and when they
In Vitro Studies of the Aurora-Kinase Inhibitor MLN837 in Prostate Cancer Cell Lines Nicole V. Julian Mentor: Ana Aparicio, M.D. Special Thanks to Brittany Kleb Department of Genitourinary Medical Oncology
Inhibition of andrographolide in RAW 264.7 murine macrophage osteoclastogenesis by downregulating the nuclear factor-kappab signaling pathway Y.Q. Ren 1 and Y.B. Zhou 2 1 Department of Orthopedics 1, Yuyao
EpiQuik Global Histone H4 Acetylation Assay Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Histone H4 Acetylation Assay Kit is suitable for specifically measuring global
FastImmune Intracellular Cytokine Staining Procedures BD has developed protocols for the detection of intracellular cytokines in activated lymphocytes and in activated monocytes. The procedures have been
Midi Plant Genomic DNA Purification Kit Cat #:DP022MD/ DP022MD-50 Size:10/50 reactions Store at RT For research use only 1 Description: The Midi Plant Genomic DNA Purification Kit provides a rapid, simple
AD Award Number: W81XWH-12-1-0248 TITLE: Targeted Inhibition of Tyrosine Kinase-Mediated Epigenetic Alterations to Prevent Resurgence of Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Dr.
RayBio Human PPAR-alpha Transcription Factor Activity Assay Kit Catalog #: TFEH-PPARa User Manual Jan 5, 2018 3607 Parkway Lane, Suite 200 Norcross, GA 30092 Tel: 1-888-494-8555 (Toll Free) or 770-729-2992,