Gypenosides Suppress Growth of Human Oral Cancer SAS Cells In Vitro and in a Murine Xenograft Model

Abstract

Purpose. Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino. The authors investigated the effects of Gyp on cell morphology, viability, cell cycle distribution, and induction of apoptosis in human oral cancer SAS cells and the determination of murine SAS xenograft model in vivo. Experimental design. Flow cytometry was used to quantify the percentage of viable cells; cell cycle distribution; sub-G1 phase (apoptosis); caspase-3, -8, and -9 activity; reactive oxygen species (ROS) production, intracellular Ca 2+ determination; and the level of mitochondrial membrane potential (ΔΨ m ). Western blotting was used to examine levels of apoptosis-associated proteins, and confocal laser microscopy was used to examine the translocation of proteins in cells. Results. Gyp induced morphological changes, decreased the percentage of viable cells, caused G0/G1 phase arrest, and triggered apoptotic cell death in SAS cells. Cell cycle arrest induced by Gyp was associated with apoptosis. The production of ROS, increased intracellular Ca 2+ levels, and the depolarization of ΔΨ m were observed. Gyp increased levels of the proapoptotic protein Bax but inhibited the levels of the antiapoptotic proteins Bcl-2 and Bcl-xl. Gyp also stimulated the release of cytochrome c and Endo G. Translocation of GADD153 to the nucleus was stimulated by Gyp. Gyp in vivo attenuated the size and volume of solid tumors in a murine xenograft model of oral cancer. Conclusions. Gyp-induced cell death occurs through caspase-dependent and caspase-independent apoptotic signaling pathways, and the compound reduced tumor size in a xenograft nu/nu mouse model of oral cancer. Materials and Methods Chemicals, Reagents, and Cell CultureGyp was kindly provided by Dr Jung-Chou Chen (Department of Chinese Medicine, China Medical University, Taichung, Taiwan). 25 Dimethyl sulfoxide (DMSO), propidium iodide (PI), potassium phosphates, ribonuclease-A, Triton X-100, Tris-HCl, and trypan blue were obtained from Sigma Chemical Co (St Louis, MO). 2,7-Dichlorodihydrofluorescein diacetate, DiOC 6 , and Fluo-3/AM were obtained from Molecular Probes/Invitrogen Corp (Eugene, OR). Dulbecco's modified Eagle's medium (DMEM), L-glutamine, fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were obtained from GIBCO BRL/Invitrogen Corp (Grand Island, NY). The SAS cell line (human oral squamous cell carcinoma) was obtained from Dr Pei-Jung Lu (Graduate Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan). Cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in 75 cm 2 tissue culture flasks at 37°C under a humidified 5% CO 2 and 95% air atmosphere as we have previously reported. 22 In Vitro StudiesAssessment of cell morphology and viability. Gyp was prepared and dissolved in DMSO. Cells (2 × 10 5 cells/well) were plated in 12-well plates in 2 mL DMEM and incubated at 37°C for 24 hours. Cells were then treated with 0, 60, 90, 120, 150, and 180 µg/mL G...

Anti-Cancerous Potential of Polysaccharide Fractions Extracted from Peony Seed Dreg on Various Human Cancer Cell Lines Via Cell Cycle Arrest and Apoptosis

“…seeds showed anti-tumor activities under in-vivo conditions (Atashrazm et al, 2016; Liu G. et al, 2016; Zheng et al, 2016; Wang et al, 2017). One latest review updated the anti-cancerous potential of gynostemma polysaccharide in different normal and transgenic cancer mice models (Hsu et al, 2011; Lu et al, 2012; Liu et al, 2014; Tai et al, 2016). Thus, in-vivo studies using tested polysaccharide fractions at the next level would generate their comprehensive efficacy report for better prediction of their anti-cancerous potential.…”

“…A recent review has reported the anti-cancerous mechanism of Gynostemma pentaphyllum (Thunb.) Makino (Jiaogulan) GPM polysaccharides through cell cycle arrest (modulation of several cell cycle regulatory proteins (CDK-2, CDK-4, and CDK-6) (Chen et al, 2009; Cheng et al, 2011), induction of apoptosis (regulation of anti-apoptotic proteins Bcl-2, Bcl-xl and pro-apoptotic proteins Bax, Bad, and Bak (Wang et al, 2002; Hsu et al, 2011; Lin et al, 2011; Lu et al, 2012), glycolysis inhibition and immunomodulation.…”

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart

“…Gypenosides (GP) are saponins of Gynostemma pentaphyllum, a traditional Chinese medicinal herb in the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated by the modern scientific methods to possess a variety of pharmaceutical properties such as anti-tumor (Lu et al 2012), antihyperlipidemia (Megalli et al 2005), and anti-hepatic fibrosis . GP can also exert cardioprotective effects in rats with induced myocardial infarction by preserving the activity of superoxide dismutase (SOD) and reducing the level of malondialdehyde (MDA) in the myocardium (Xiong et al 1990).…”

Gypenosides Synergistically Enhances the Anti-Tumor Effect of 5-Fluorouracil on Colorectal Cancer In Vitro and In Vivo: A Role for Oxidative Stress-Mediated DNA Damage and p53 Activation

“…Gypenosides (Gyp), the major components extracts from Gynostemma pentaphyllum (Thunb.) Makino, exists mainly as dammarane-type triterpene glycosides ( Fig 1A ) and has been used as a traditional popular folk medicine in Asia for centuries to treat cancer [ 11 – 13 ], hyperlipoproteinemia [ 14 ], hepatitis [ 15 ], and cardiovascular disease [ 16 ]. Experimental study has reported that Gyp could trigger apoptosis in human colorectal cancer 205 cells through mitochondria-dependent pathway and activation of caspase-3 [ 17 ].…”