Wang et al. BMC Complementary and Alternative Medicine 2013, 13:2 http://www.biomedcentral.com/1472-6882/13/2 RESEARCH ARTICLE Open Access Paraptosis and NF-κB activation are associated with protopanaxadiol-induced cancer chemoprevention Chong-Zhi Wang1,2, Binghui Li3, Xiao-Dong Wen1,2, Zhiyu Zhang1,2, Chunhao Yu1,2, Tyler D Calway1,2, Tong-Chuan He4, Wei Du3 and Chun-Su Yuan1,2,5,6* Abstract Background: Protopanaxadiol (PPD) is a triterpenoid that can be prepared from steamed ginseng. PPD possesses anticancer potential via caspase-dependent apoptosis. Whether paraptosis, a type of the caspase-independent cell death, is also induced by PPD has not been evaluated. Methods: Cell death, the cell cycle and intracellular reactive oxygen species (ROS) were analyzed by flow cytometry after staining with annexin V/PI, PI/RNase or H2DCFDA. We observed morphological changes by crystal violet staining assay. Mitochondrial swelling was measured by ultraviolet–visible spectrophotometry. The activation of NF-κB was measured by luciferase reporter assay. Results: At comparable concentrations of 5-fluorouracil, PPD induced more cell death in human colorectal cancer cell lines HCT-116 and SW-480. We demonstrated that PPD induced paraptosis in these cancer cells. PPD treatment significantly increased the percentage of cancer cells with cytoplasmic vacuoles. After the cells were treated with PPD and cycloheximides, cytoplasmic vacuole generation was inhibited. The paraptotic induction effect of PPD was also supported by the results of the mitochondrial swelling assay. PPD induced ROS production in cancer cells, which activated the NF-κB pathway. Blockage of ROS by NAC or PS-1145 inhibited the activation of NF-κB signaling. Conclusions: PPD induces colorectal cancer cell death in part by induction of paraptosis. The anticancer activity of PPD may be enhanced by antioxidants such as green tea, which also inhibit the activation of NF-κB signaling. Keywords: Ginseng, Protopanaxadiol, PPD, Paraptosis, Cytoplasmic vacuoles, Mitochondrial swelling, Antioxidant, Cancer chemoprevention Background option for cancer chemoprevention and new drug deve- The clinical management of cancer invariably involves lopment [6-8]. diverse conventional modalities, including surgery, radia- Long-term consumption of certain botanicals, such as tion, and chemotherapy [1]. Because of the complexity ginseng, is associated with a reduction in cancer inci- of human cancer, alternative management may be nee- dence in humans [9,10]. Anticancer potential has been ded to improve the efficacy of therapeutic treatments observed with ginseng and its compounds, including the and the quality of life of patients [2]. Cancer chemo- enhancement of 5-fluoruracil’s anti-proliferative effects prevention or treatment may combine natural products on human cancer cells [11-13]. Steaming ginseng changes with chemotherapeutic agents to inhibit tumor develop- its ginsenoside profile and increases its anticancer poten- ment [3-5]. Natural products have been shown to be one tial [14,15]. The ginsenoside Rh2 in ginseng induced apoptosis and * Correspondence: [email protected] paraptosis-like cell death in colon cancer cells [16]. Gin- 1 Tang Center for Herbal Medicine Research, University of Chicago, Chicago, senoside Rh2 levels are low in untreated ginseng, but IL, USA 2Department of Anesthesia and Critical Care, University of Chicago, Chicago, after steaming, Rh2 levels increase [17]. In a recent review IL, USA of the relationship between the structure and function of Full list of author information is available at the end of the article © 2013 Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wang et al. BMC Complementary and Alternative Medicine 2013, 13:2 Page 2 of 8 http://www.biomedcentral.com/1472-6882/13/2 ginsenosides, we proposed that reducing sugar molecules antioxidant dietary supplements are often self-administered in ginsenosides increases their anticancer bioactivity [7]. by cancer patients [20,21]. The present study data suggest We also observed that further heat treatment of ginseng that paraptosis and NF-κB activation are associated with initiated the conversion of Rh2 to protopanaxadiol (PPD). PPD-induced cancer chemoprevention. In this transformation, another sugar molecule is removed from Rh2 (see Discussion and Figure 1). Methods PPD possesses anticancer potential because it induces Chemicals and reagents cell apoptosis, a programmed cell death that is caspase- DMSO and other solvents were obtained from Fisher Sci- dependent [18]. Paraptosis, another type of cell death, is entific (Pittsburgh, PA). Trypsin, McCoy’s 5A, Leibovitz’s characterized by the accumulation of cytoplasmic vacuoles L-15 medium, fetal bovine serum (FBS), and penicillin/ and mitochondrial swelling [19]. Whether PPD-induced streptomycin solution (200×) were obtained from Media- cell death also is mediated by caspase-independent para- tech, Inc. (Herndon, VA). N-Acetyl-L-cysteine (NAC), PS- ptosis, like Rh2, is not known. In previous studies, Rh2 1145, propidium iodide (PI) and RNase were obtained increased levels of reactive oxygen species (ROS) and acti- from Sigma (St. Louis, MO). NAC, which is an antio- vated the NF-κB survival pathway [16]. It would be inter- xidant, was dissolved in the growth medium. PS-1145, a esting to know whether ROS blockage and inhibition of specific inhibitor of the NF-κB pathway, was dissolved NF-κB signaling increases PPD-induced cell death and in DMSO as a 20 mM stock buffer. Protopanaxadiol whether PPD’s effect is enhanced by antioxidants because (PPD) was obtained from National Institutes for Food Figure 1 Transformation pathways of panaxadiol group ginsenosides Rb1, Rc and Rd by steaming treatment and intestinal microbiota. During steaming, ginsenosides Rb1, Rc and Rd are mainly transformed to Rg5, Rk1, and Rg3. Rg3 then converts to Rh2 and PPD. In addition, intestinal microbiota metabolize Rb1, Rc and Rd to compound K, which can be further converted to PPD (dotted lines). Wang et al. BMC Complementary and Alternative Medicine 2013, 13:2 Page 3 of 8 http://www.biomedcentral.com/1472-6882/13/2 and Drug Control (Beijing, China). 5-Fluorouracil (5-FU) stained with 0.2% crystal violet in 10% phosphate-buffered was obtained from American Pharmaceutical Partners formaldehyde for 2 min. The staining solution was remo- Inc. (Schaumburg, IL). Luciferase assay kits were obtained ved and the cells were washed twice with PBS. The re- from Promega (Madison, WI). Annexin V Apoptosis Kit maining cells adhering to the wells were observed under was purchased from BD Biosciences (San Diego, CA). Re- the microscope and photographed. 0 0 active oxygen species (ROS) dye 2 ,7 -dichlorodihydro- fluorescein diacetate (H2DCFDA) and L-glutamine were obtained from Invitrogen (Carlsbad, CA). Mitochondrial swelling assay Human liver mitochondria were obtained from Xeno- Cell culture tech LLC (Lenexa, KS). Before the experiment, the mito- Human colorectal cancer cells HCT-116 and SW-480 chondria were suspended in 230 mM mannitol, 70 mM – were obtained from the American Type Culture Collec- sucrose, 10 mM Tris HCl, and 1 mM EDTA, with a tion (ATCC, Manassas, VA), and were maintained in protein concentration of 0.5 mg/ml. After exposure to μ McCoy’s 5A (HCT-116) or Leibovitz’s L-15 (SW-480) 35 M of PPD or control condition at 25°C, mitochon- – medium supplemented with 5% fetal bovine serum, 50 IU drial swelling was measured by the ultraviolet visible of penicillin/streptomycin and 2 mmol/L of L-glutamine spectrophotometry method. The absorbance changes at 540 nm were monitored [22]. in a humidified atmosphere with 5% CO2 at 37°C. Cell death assay Intracellular ROS assay Cells were seeded into 24-well plates (2 × 105 cells/well). Intracellular ROS production was monitored by the per- Samples were prepared based on the instruction pro- meable fluorescence dye, H2DCFDA. H2DCFDA can rea- vided with the Annexin V Apoptosis Kit. Briefly, after dily react with ROS to form the fluorescent product treatment as indicated in the result section, the adhe- 2,7-dichlorofluorescein (DCF). The intracellular fluores- rent and detached cells were collected and washed twice cence intensity of DCF is proportional to the amount of with binding buffer containing 10 mM HEPES, pH 7.4, ROS generated by the cells. After the indicated treatment, 140 mM NaCl, 2.5 mM CaCl, and then 1 × 105 cells were the cells were incubated with 10 μM of H2DCFDA for resuspended in 100 μl of binding buffer. 5 μl of annexin thirty minutes and then cells were harvested and resus- V-FITC and 10 μl of propidium iodide (50 μg/ml, stocking pended in PBS (106 cells/mL). The fluorescence intensity concentration) were added to the cell suspension. After of intracellular DCF (excitation 488 nm, emission 530 nm) gently mixing, the cells were incubated for 15 min at was measured using a FACScan flow cytometer. room temperature, and then 400 μl of binding buffer was added to get the sample ready. Quantification of cell death was performed using a FACScan flow cytome- Luciferase activity assay ter (BD Biosciences, San Jose, CA). All the data analyses The plasmids containing the luciferase reporter gene with were performed using FlowJo analysis software, version or without a NF-κB response element and phRL-TK plas- 6.0 (FlowJo LLC, Ashland, OR). Annexin V-positive and/ mid for the transfection control were donated by Liao’s or PI-positive cells were considered as cell death. lab (Ben May Department for Cancer Research, University of Chicago). 104 cells were seeded into 48-well plates for Cell cycle analysis 24 h and were co-transfected with 0.5 μg of plasmid con- 5 Cells were seeded in 24-well plates (2 × 10 cells/well). taining report construct and 10 ng of phRL-TK using After 48 h of drug exposure or control conditions, cells transfection reagent Effectene (Qiagen, Chatsworth, CA).