Paraptosis and NF-κB activation are associated with protopanaxadiol-induced cancer chemoprevention
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 4 October 2012
Accepted: 19 December 2012
Published: 3 January 2013
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.
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.
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.
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.
KeywordsGinseng Protopanaxadiol PPD Paraptosis Cytoplasmic vacuoles Mitochondrial swelling Antioxidant Cancer chemoprevention
The clinical management of cancer invariably involves diverse conventional modalities, including surgery, radiation, and chemotherapy . Because of the complexity of human cancer, alternative management may be needed to improve the efficacy of therapeutic treatments and the quality of life of patients . Cancer chemoprevention or treatment may combine natural products with chemotherapeutic agents to inhibit tumor development [3–5]. Natural products have been shown to be one option for cancer chemoprevention and new drug development [6–8].
Long-term consumption of certain botanicals, such as ginseng, is associated with a reduction in cancer incidence in humans [9, 10]. Anticancer potential has been observed with ginseng and its compounds, including the enhancement of 5-fluoruracil’s anti-proliferative effects on human cancer cells [11–13]. Steaming ginseng changes its ginsenoside profile and increases its anticancer potential [14, 15].
PPD possesses anticancer potential because it induces cell apoptosis, a programmed cell death that is caspase-dependent . Paraptosis, another type of cell death, is characterized by the accumulation of cytoplasmic vacuoles and mitochondrial swelling . Whether PPD-induced cell death also is mediated by caspase-independent paraptosis, like Rh2, is not known. In previous studies, Rh2 increased levels of reactive oxygen species (ROS) and activated the NF-κB survival pathway . It would be interesting to know whether ROS blockage and inhibition of NF-κB signaling increases PPD-induced cell death and whether PPD’s effect is enhanced by antioxidants because antioxidant dietary supplements are often self-administered by cancer patients [20, 21]. The present study data suggest that paraptosis and NF-κB activation are associated with PPD-induced cancer chemoprevention.
Chemicals and reagents
DMSO and other solvents were obtained from Fisher Scientific (Pittsburgh, PA). Trypsin, McCoy’s 5A, Leibovitz’s L-15 medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (200×) were obtained from Mediatech, Inc. (Herndon, VA). N-Acetyl-L-cysteine (NAC), PS-1145, propidium iodide (PI) and RNase were obtained from Sigma (St. Louis, MO). NAC, which is an antioxidant, was dissolved in the growth medium. PS-1145, a specific inhibitor of the NF-κB pathway, was dissolved in DMSO as a 20 mM stock buffer. Protopanaxadiol (PPD) was obtained from National Institutes for Food and Drug Control (Beijing, China). 5-Fluorouracil (5-FU) was obtained from American Pharmaceutical Partners Inc. (Schaumburg, IL). Luciferase assay kits were obtained from Promega (Madison, WI). Annexin V Apoptosis Kit was purchased from BD Biosciences (San Diego, CA). Reactive oxygen species (ROS) dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and L-glutamine were obtained from Invitrogen (Carlsbad, CA).
Human colorectal cancer cells HCT-116 and SW-480 were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and were maintained in McCoy’s 5A (HCT-116) or Leibovitz’s L-15 (SW-480) medium supplemented with 5% fetal bovine serum, 50 IU of penicillin/streptomycin and 2 mmol/L of L-glutamine in a humidified atmosphere with 5% CO2 at 37°C.
Cell death assay
Cells were seeded into 24-well plates (2 × 105 cells/well). Samples were prepared based on the instruction provided with the Annexin V Apoptosis Kit. Briefly, after treatment as indicated in the result section, the adherent and detached cells were collected and washed twice with binding buffer containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl, and then 1 × 105 cells were resuspended in 100 μl of binding buffer. 5 μl of annexin V-FITC and 10 μl of propidium iodide (50 μg/ml, stocking concentration) were added to the cell suspension. After gently mixing, the cells were incubated for 15 min at 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 cytometer (BD Biosciences, San Jose, CA). All the data analyses were performed using FlowJo analysis software, version 6.0 (FlowJo LLC, Ashland, OR). Annexin V-positive and/or PI-positive cells were considered as cell death.
Cell cycle analysis
Cells were seeded in 24-well plates (2 × 105 cells/well). After 48 h of drug exposure or control conditions, cells were fixed gently by adding 80% ethanol before they were placed in a freezer for 2 h; then cells were treated with 0.25% Triton X-100 for 5 min in an ice bath. The cells were resuspended in 130 μl of PBS. Then, 5 μl of PI/RNase staining solution was added to the cell suspension. Cells were incubated in a dark room for 10 min at room temperature and analyzed using a FACScan flow cytometer.
Crystal violet staining assay
HCT-116 cells were seeded in 24-well plates (1 × 105 cells/well). After 12 h of drug exposure or control conditions, the medium was removed and the cells were washed and stained with 0.2% crystal violet in 10% phosphate-buffered formaldehyde for 2 min. The staining solution was removed and the cells were washed twice with PBS. The remaining cells adhering to the wells were observed under the microscope and photographed.
Mitochondrial swelling assay
Human liver mitochondria were obtained from Xenotech LLC (Lenexa, KS). Before the experiment, the mitochondria were suspended in 230 mM mannitol, 70 mM sucrose, 10 mM Tris–HCl, and 1 mM EDTA, with a protein concentration of 0.5 mg/ml. After exposure to 35 μM of PPD or control condition at 25°C, mitochondrial swelling was measured by the ultraviolet–visible spectrophotometry method. The absorbance changes at 540 nm were monitored .
Intracellular ROS assay
Intracellular ROS production was monitored by the permeable fluorescence dye, H2DCFDA. H2DCFDA can readily react with ROS to form the fluorescent product 2,7-dichlorofluorescein (DCF). The intracellular fluorescence intensity of DCF is proportional to the amount of ROS generated by the cells. After the indicated treatment, the cells were incubated with 10 μM of H2DCFDA for thirty minutes and then cells were harvested and resuspended in PBS (106 cells/mL). The fluorescence intensity of intracellular DCF (excitation 488 nm, emission 530 nm) was measured using a FACScan flow cytometer.
Luciferase activity assay
The plasmids containing the luciferase reporter gene with or without a NF-κB response element and phRL-TK plasmid for the transfection control were donated by Liao’s lab (Ben May Department for Cancer Research, University of Chicago). 104 cells were seeded into 48-well plates for 24 h and were co-transfected with 0.5 μg of plasmid containing report construct and 10 ng of phRL-TK using transfection reagent Effectene (Qiagen, Chatsworth, CA). At 24 h post-transfection, the cells were treated as desired. Luciferase activity was measured with a commercial kit (Promega Dual luciferase II) on a Monolight luminometer (Becton Dickinson, Franklin Lakes, NJ).
All experiments were performed in triplicate. The data are presented as mean ± standard error (SE). A one-way ANOVA determined whether the results had statistical significance. In some cases, a Student’s t-test was used for comparing two groups. The level of statistical significance was set at P < 0.05.
Effects of PPD on cell death and cell cycle of human colorectal cancer cells
PPD-induced paraptosis-like cell death
Effects of PPD on induction of ROS
PPD activated the NF-κB pathway linked to ROS generation
Decreasing the level of ROS in the cell should decrease NF-κB reporter activity, assuming that increased levels of ROS are required to induce NF-κB signaling. Addition of the antioxidant NAC significantly blocked the PPD-mediated induction of NF-κB transcriptional activity (Figure 6B) and increased cell death in HCT-116 cells, suggesting that PPD-induced ROS contribute to the activation of the NF-κB pathway.
We expected that the activated NF-κB pathway would counteract PPD-induced cell death in colorectal cancer cells because a decrease in ROS decreases NF-κB transcriptional activity. To further test the relationship of PPD-induced NF-κB activation and cell death, we inhibited the NF-κB pathway by adding PS-1145, a specific inhibitor of the NF-κB pathway, to PPD-treated HCT-116 cells. PS-1145 significantly inhibited both basal and PPD-induced NF-κB transcriptional activity (Figure 6C), and increased PPD-induced cell death in HCT-116 cells. PPD-induced ROS contributed to the survival of colorectal cancer cells via activation of the NF-κB pathway. Therefore, PPD-induced colorectal cancer cell death is enhanced by antioxidants or an NF-κB inhibitor.
After several years of cultivation in China or Korea, the root of Asian ginseng is harvested and steamed at standard boiling temperature; the steamed root is called red ginseng. It is believed that the steaming changes the ginsenoside composition profile, improving pharmacological activities. Our group used controlled variable steaming parameters to treat American ginseng and notoginseng to obtain desired ginsenoside profiles for increasing cancer chemopreventive effects [14, 15].
American ginseng is a commonly used dietary supplement in United States. The major ginsenosides in American ginseng belong to the protopanaxadiol group and include Rb1, Rc and Rd . Ginsenoside Rb1 is the most abundant ginsenoside in American ginseng, accounting for over 50% of total ginsenosides in many samples. Figure 1 elucidates the ginsenoside transformation by steaming treatment and intestinal microbiota. During steaming treatment, the protopanaxadiol group of ginsenosides are mainly transformed via three pathways: 1) to Rg5 and then to Rh4; 2) to Rk1 and then to Rk3; and 3) a major route, to Rg3, Rh2, and then to PPD [15, 24]. Interestingly, after oral ginseng administration, gut microbiota, which normally exist in the gastrointestinal tract, can convert Rb1 and other protopanaxadiol group ginsenosides into their metabolite, ginsenoside compound K (CK) [25, 26]. Recently, our study data showed that a relatively high amount of CK could be transformed to PPD by gut microbiota (unpublished data). Thus, the evaluation of cancer chemopreventive effects of PPD and its related mechanism of action is particularly pertinent to ginseng anticancer investigations.
Two representative human cancer cell lines, HCT-116 and SW-480, which differ in the expression of the p53 tumor suppressor gene, were used in our cell death observation. HCT-116 is p53 wild-type, while SW-480 has mutated p53. Tumor suppressor gene p53 is thought to be important in the cellular response to chemotherapeutic agent-induced damage. Mutation in p53 has been shown to correlate with an increased resistance to chemotherapy in cancer cells . Our results showed that PPD significantly induced cell death in these two cell lines in a concentration-dependent manner, and HCT-116 was more sensitive even at lower concentrations. After PPD treatment, accumulation of cytoplasmic vacuoles was observed in the cancer cells, suggesting that paraptosis, another type of programmed cell death , was associated with PPD’s effect. Paraptosis is further supported by a mitochondrial swelling assay and by the observation that PPD used in combination with cycloheximides, a protein synthesis inhibitor, blocked PPD-induced vacuole formation.
5-Fluorouracil (5-FU) is a chemotherapeutic agent widely used for cancers of different organs including colorectum . The clinical response rate of 5-FU in colorectal cancer treatment was 20-30%, and in combination with irinotecan or oxaliplatin, up to 50% [29, 30]. Consistent with others , we observed that the cell death induction of 5-FU is not very impressive, with apoptosis as a main cellular response to treatment with 5-FU. At comparable concentrations, our data showed that PPD more significantly induced cell death. It is crucial for future work to identify combinatory therapies of natural product-derived compounds to increase the effectiveness of 5-FU on human colorectal cancer.
Our data showed that in both tested colon cancer cell lines, PPD significantly induced ROS production. Further, PPD activated the NF-κB pathway, which is linked to ROS generation. When the antioxidant NAC was used, NF-κB reporter activity levels were significantly reduced. This observation suggested that co-administration of PPD and a botanical antioxidant may achieve better cancer chemopreventive effects. Panaxadiol (PD) is a purified sapogenin of ginseng saponins, which has exhibited significant anticancer activity . Structurally, PD is very similar to PPD, with basically only side chain cyclization . Epigallocatechin gallate (EGCG), a major catechin in green tea, is a strong botanical antioxidant. Recently, we reported synergistic anticancer effects of PD and EGCG . Based on the similar chemical structure of PD and PPD, it would be expected that co-administration of PPD and EGCG could also initiate increased cancer chemoprevention activities.
Results from the present study suggest that paraptosis and NF-κB activation are associated with PPD-induced cancer chemoprevention. The activation of PPD’s NF-κB pathway is linked to its increased ROS level.
This work was supported in part by the NIH/NCCAM AT004418 and AT005362, NIH GM074197 and 5P30DK042086, NIH/NCI CA149275, and DOD W81XWH-10-1-0077.
- Wood CG: Multimodal approaches in the management of locally advanced and metastatic renal cell carcinoma: combining surgery and systemic therapies to improve patient outcome. Clin Cancer Res. 2007, 13 (2 Pt 2): 697s-702s.View ArticlePubMedGoogle Scholar
- Oettle H, Post S, Neuhaus P, Gellert K, Langrehr J, Ridwelski K, Schramm H, Fahlke J, Zuelke C, Burkart C, Gutberlet K, Kettner E, Schmalenberg H, Weigang-Koehler K, Bechstein WO, Niedergethmann M, Schmidt-Wolf I, Roll L, Doerken B, Riess H: Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. JAMA. 2007, 297 (3): 267-277. 10.1001/jama.297.3.267.View ArticlePubMedGoogle Scholar
- Biagi JJ, Raphael MJ, Mackillop WJ, Kong W, King WD, Booth CM: Association between time to initiation of adjuvant chemotherapy and survival in colorectal cancer: a systematic review and meta-analysis. JAMA. 2011, 305 (22): 2335-2342. 10.1001/jama.2011.749.View ArticlePubMedGoogle Scholar
- Wang CZ, Calway T, Yuan CS: Herbal medicines as adjuvants for cancer therapeutics. Am J Chin Med. 2012, 40 (4): 657-669. 10.1142/S0192415X12500498.View ArticlePubMedPubMed CentralGoogle Scholar
- Bougnoux P, Hajjaji N, Maheo K, Couet C, Chevalier S: Fatty acids and breast cancer: sensitization to treatments and prevention of metastatic re-growth. Prog Lipid Res. 2010, 49 (1): 76-86. 10.1016/j.plipres.2009.08.003.View ArticlePubMedGoogle Scholar
- Lee KH: Discovery and development of natural product-derived chemotherapeutic agents based on a medicinal chemistry approach. J Nat Prod. 2010, 73 (3): 500-516. 10.1021/np900821e.View ArticlePubMedPubMed CentralGoogle Scholar
- Qi LW, Wang CZ, Yuan CS: American ginseng: potential structure-function relationship in cancer chemoprevention. Biochem Pharmacol. 2010, 80 (7): 947-954. 10.1016/j.bcp.2010.06.023.View ArticlePubMedGoogle Scholar
- Xu Z, Chen X, Zhong Z, Chen L, Wang Y: Ganoderma lucidum polysaccharides: immunomodulation and potential anti-tumor activities. Am J Chin Med. 2011, 39 (1): 15-27. 10.1142/S0192415X11008610.View ArticlePubMedGoogle Scholar
- Yun TK: Panax ginseng–a non-organ-specific cancer preventive?. Lancet Oncol. 2001, 2 (1): 49-55. 10.1016/S1470-2045(00)00196-0.View ArticlePubMedGoogle Scholar
- Yun TK, Zheng S, Choi SY, Cai SR, Lee YS, Liu XY, Cho KJ, Park KY: Non-organ-specific preventive effect of long-term administration of Korean red ginseng extract on incidence of human cancers. J Med Food. 2010, 13 (3): 489-494. 10.1089/jmf.2009.1275.View ArticlePubMedGoogle Scholar
- Li XL, Wang CZ, Mehendale SR, Sun S, Wang Q, Yuan CS: Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells. Cancer Chemother Pharmacol. 2009, 64 (6): 1097-1104. 10.1007/s00280-009-0966-0.View ArticlePubMedGoogle Scholar
- Fishbein AB, Wang CZ, Li XL, Mehendale SR, Sun S, Aung HH, Yuan CS: Asian ginseng enhances the anti-proliferative effect of 5-fluorouracil on human colorectal cancer: comparison between white and red ginseng. Arch Pharm Res. 2009, 32 (4): 505-513. 10.1007/s12272-009-1405-9.View ArticlePubMedGoogle Scholar
- Hwang JW, Oh JH, Yoo HS, Lee YW, Cho CK, Kwon KR, Yoon JH, Park J, Her S, Lee ZW, Jang IS, Choi JS: Mountain ginseng extract exhibits anti-lung cancer activity by inhibiting the nuclear translocation of NF-kappaB. Am J Chin Med. 2012, 40 (1): 187-202. 10.1142/S0192415X12500152.View ArticlePubMedGoogle Scholar
- Wang CZ, Aung HH, Ni M, Wu JA, Tong R, Wicks S, He TC, Yuan CS: Red American ginseng: ginsenoside constituents and antiproliferative activities of heat-processed Panax quinquefolius roots. Planta Med. 2007, 73 (7): 669-674. 10.1055/s-2007-981524.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun S, Qi LW, Du GJ, Mehendale SR, Wang CZ, Yuan CS: Red notoginseng: higher ginsenoside content and stronger anticancer potential than Asian and American ginseng. Food Chem. 2011, 125 (4): 1299-1305. 10.1016/j.foodchem.2010.10.049.View ArticlePubMedPubMed CentralGoogle Scholar
- Li B, Zhao J, Wang CZ, Searle J, He TC, Yuan CS, Du W: Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53. Cancer Lett. 2011, 301 (2): 185-192. 10.1016/j.canlet.2010.11.015.View ArticlePubMedGoogle Scholar
- Wang CZ, Zhang B, Song WX, Wang A, Ni M, Luo X, Aung HH, Xie JT, Tong R, He TC, Yuan CS: Steamed American ginseng berry: ginsenoside analyses and anticancer activities. J Agric Food Chem. 2006, 54 (26): 9936-9942. 10.1021/jf062467k.View ArticlePubMedGoogle Scholar
- Du GJ, Dai Q, Williams S, Wang CZ, Yuan CS: Synthesis of protopanaxadiol derivatives and evaluation of their anticancer activities. Anticancer Drugs. 2011, 22 (1): 35-45. 10.1097/CAD.0b013e32833fde29.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen TS, Wang XP, Sun L, Wang LX, Xing D, Mok M: Taxol induces caspase-independent cytoplasmic vacuolization and cell death through endoplasmic reticulum (ER) swelling in ASTC-a-1 cells. Cancer Lett. 2008, 270 (1): 164-172. 10.1016/j.canlet.2008.05.008.View ArticlePubMedGoogle Scholar
- Greenlee H, Gammon MD, Abrahamson PE, Gaudet MM, Terry MB, Hershman DL, Desai M, Teitelbaum SL, Neugut AI, Jacobson JS: Prevalence and predictors of antioxidant supplement use during breast cancer treatment: the Long Island Breast Cancer Study Project. Cancer. 2009, 115 (14): 3271-3282. 10.1002/cncr.24378.View ArticlePubMedPubMed CentralGoogle Scholar
- Myung SK, Kim Y, Ju W, Choi HJ, Bae WK: Effects of antioxidant supplements on cancer prevention: meta-analysis of randomized controlled trials. Ann Oncol. 2010, 21 (1): 166-179. 10.1093/annonc/mdp286.View ArticlePubMedGoogle Scholar
- Fernandez-Gomez FJ, Galindo MF, Gomez-Lazaro M, Gonzalez-Garcia C, Cena V, Aguirre N, Jordan J: Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions. Neuroscience. 2005, 133 (4): 959-967. 10.1016/j.neuroscience.2005.03.019.View ArticlePubMedGoogle Scholar
- Qi LW, Wang CZ, Yuan CS: Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry. 2011, 72 (8): 689-699. 10.1016/j.phytochem.2011.02.012.View ArticlePubMedPubMed CentralGoogle Scholar
- Qi LW, Wang CZ, Yuan CS: Isolation and analysis of ginseng: advances and challenges. Nat Prod Rep. 2011, 28 (3): 467-495. 10.1039/c0np00057d.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang CZ, Kim KE, Du GJ, Qi LW, Wen XD, Li P, Bauer BA, Bissonnette MB, Musch MW, Chang EB, Yuan CS: Ultra-performance liquid chromatography and time-of-flight mass spectrometry analysis of ginsenoside metabolites in human plasma. Am J Chin Med. 2011, 39 (6): 1161-1171. 10.1142/S0192415X11009470.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang HY, Qi LW, Wang CZ, Li P: Bioactivity enhancement of herbal supplements by intestinal microbiota focusing on ginsenosides. Am J Chin Med. 2011, 39 (6): 1103-1115. 10.1142/S0192415X11009433.View ArticlePubMedPubMed CentralGoogle Scholar
- Din FV, Dunlop MG, Stark LA: Evidence for colorectal cancer cell specificity of aspirin effects on NF kappa B signalling and apoptosis. Br J Cancer. 2004, 91 (2): 381-388.PubMedPubMed CentralGoogle Scholar
- Longley DB, Harkin DP, Johnston PG: 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003, 3 (5): 330-338. 10.1038/nrc1074.View ArticlePubMedGoogle Scholar
- Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, Jandik P, Iveson T, Carmichael J, Alakl M, Gruia G, Awad L, Rougier P: Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet. 2000, 355 (9209): 1041-1047. 10.1016/S0140-6736(00)02034-1.View ArticlePubMedGoogle Scholar
- Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, Chollet P, Llory JF, Letourneau Y, Coudert B, Bertheaut-Cvitkovic F, Larregain-Fournier D, Le Rol A, Walter S, Adam R, Misset JL, Levi F: Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2000, 18 (1): 136-147.PubMedGoogle Scholar
- Choudhary B, Hanski ML, Zeitz M, Hanski C: Proliferation rate but not mismatch repair affects the long-term response of colon carcinoma cells to 5FU treatment. Cancer Lett. 2012, 320 (1): 56-64. 10.1016/j.canlet.2012.01.025.View ArticlePubMedGoogle Scholar
- Du GJ, Wang CZ, Qi LW, Zhang ZY, Calway T, He TC, Du W, Yuan CS: The synergistic apoptotic interaction of panaxadiol and epigallocatechin gallate in human colorectal cancer cells. Phytother Res. 2012, 26: 10.1002/ptr.4707. In pressGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/2/prepub
This article is published under license to 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.