TA extracts are mainly composed of coumarins, alkaloids, benzenoids, and their derivatives ( Hu et al., 2014). Modern pharmacologic researches have confirmed that TA extracts have multiple biological activities, including anti-arthritis ( Yang et al., 2013), anti-inflammatory ( Hao et al., 2004 Balasubramaniam et al., 2011 Kariuki et al., 2013 Tong et al., 2014), anti-microbial ( Narod et al., 2004 Duraipandiyan and Ignacimuthu, 2009 Karunai Raj et al., 2012), anti-parasitic ( Shan et al., 2014), anti-oxidant ( Balasubramaniam et al., 2011 Irudayaraj et al., 2012 Stephen Irudayaraj et al., 2012 Ceballos et al., 2013), anti-platelet ( Tsai et al., 1998), anti-malarial ( Gakunju et al., 1995 Oketch-Rabah et al., 2000), anti-diabetic ( Irudayaraj et al., 2012), anti-tumor ( Iwasaki et al., 2006), and analgesic ( Hao et al., 2004 Kimang’a et al., 2016). (Rutaceae) (TA) has been widely used as traditional Chinese medicine for the treatment of various diseases in China ( Yang et al., 2013 Tong et al., 2014). Taken together, these results indicated that DF induced cell cycle arrest at G2/M phase and apoptosis in HT-29 cells, and could be a promising source for developing natural therapeutics for colon cancer. Furthermore, we found that HT-29 cell cycle arrest induced by DF could be the result of reactive oxygen species (ROS), as the ROS scavenger N-acetyl cysteine (NAC) attenuating it. DF also induced phosphatidylserine externalization and activated caspases -8, -9, and -3, suggesting DF induced apoptosis through intrinsic and extrinsic pathways. As a result, the dichloromethane fraction (DF) was found to possess the highest anti-proliferative activity with IC 50 value at 18 μg/mL among all of the four extracts from TA, and strongly inhibited HT-29 cell growth and halted cell cycle progression in G2/M phase. Therefore, we firstly evaluated the effects of different extracts of TA on the growth of human colon cancer cells, and then tried to further elucidate their underlying molecular mechanisms. However, the anti-cancer effects and the action mechanisms of TA remain elusive. (TA) has been often used in Chinese folk medicine to treat different diseases, including but not limited to arthritis, injuries, stomachache, and even tumors. 4Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, China.3Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.2University of Chinese Academy of Sciences, Beijing, China.1Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China.Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.Xun Li 1,2 Zidong Qiu 1,2 Qinghao Jin 1,3 Guilin Chen 1,4 Mingquan Guo 1,4* PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains.Ĭells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187.
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