Elsevier

Oral Oncology

Volume 45, Issue 3, March 2009, Pages 273-283
Oral Oncology

Gypenosides induced G0/G1 arrest via CHk2 and apoptosis through endoplasmic reticulum stress and mitochondria-dependent pathways in human tongue cancer SCC-4 cells

https://doi.org/10.1016/j.oraloncology.2008.05.012Get rights and content

Summary

Gypenosides (Gyp), a component of Gynostemma pentaphyllum Makino, was selected for examining the effects on the cell viability, cell cycle and induction of apoptosis in human tongue cancer SCC-4 cells. Gyp induced cytotoxicity (decreased the percentage of viable cells) in SCC-4 cells appeared to be associated with induction of cell cycle arrest (G0/G1 arrest), apoptotic cell death based on Gyp induced morphological changes and DNA fragmentation and increased the sub-G1 group in examined SCC-4 cells. The production of reactive oxygen species and Ca2+ and the depolarization of mitochondrial membrane potential were observed, dose- and time-dependently, after treatment of SCC-4 cells with various concentrations of Gyp. Gyp inhibited the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xl, but promoted the levels of the pro-apoptotic protein Bax. Western blotting showed the releases of cytochrome c and Endo G and both were also confirmed by confocal laser microscopic systems. The GADD153 moved to nuclei (nuclear translocation). In conclusion, Gyp induced ER stress and production of reactive oxygen species and Ca2+, change the ratio of Bcl-2 and Bax, followed by the dysfunction of mitochondria, caused cytochrome c release, activation of caspase-3 before leading to apoptosis. These results provide information towards an understanding of the mechanisms by which Gyp induces cell cycle arrest and apoptosis in human tongue cancer cells.

Introduction

Many anti-cancer agents have been obtained from natural plants and their derivatives, in particular, paclitaxel which is derived from Taxus brevifolia1, 2 and it has been used clinically for over twenty years. Gypenosides (Gyp) are the major components in Gynostemma pentaphyllum Makino that has been the popular folk medicine in the Chinese population for centuries. Many reports have demonstrated that Gyp can be used for treatment of hepatitis,3 hyperlipoproteinemia,4, 5 cardiovascular disease6 and cancer.7 Gyp has been shown to have anti-inflammatory,8 anti-thrombotic,9 anti-oxidative8 and anti-cancer10, 11, 12, 13 actions. Treatment of human hepatoma cells with Gyp induced apoptosis through the up-regulation of Bax and Bak, the down-regulation of Bcl-2, release of mitochondrial cytochrome c and activation of the caspase cascade.12 In our laboratory, we also found out that Gyp induced apoptosis in human colon cancer cells through the mitochondria-dependent pathways and activation of caspase-3.14 Earlier studies also showed that Gyp affected N-acetyltransferase activity and gene expression in human cervical cancer cells.15 Recently, it was reported that Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain’s microsomal Na(+), K(+)-ATPase activities in rats.16

Oral and oropharyngeal cancers are one of the health problems worldwide and accounting for over 300,000 cases annually,17 is more common among males than females.17 Tobacco and alcohol consumption have been reported to be the major factors for the development of oral cancers18, 19, 20 but diets low in carotenoids and vitamin A, poor oral hygiene and indoor air pollution are also recognized to be the factors for oral cancer.21, 22, 23, 24 However, betel quid chewing is one of the important factors of oral cancer in Taiwan. There is no available information to address the effects of Gyp on human tongue cancer cells. Therefore, to understand the effects of Gyp on human tongue cancer cells, we selected the human tongue cancer SCC-4 cells for examining the mechanisms underlying the induction of cell cycle arrest and apoptosis, with particular focus on the production of reactive oxygen species (ROS), Ca2+, endoplasm reticulum stress and mitochondrial membrane potential, the expressions of GADD 153, GRP78, Bcl-2, Bax, cytochrome c, p53 and the activation of caspase-3, -8 and -12, with the resulting DNA fragmentation.

Section snippets

Chemicals, reagents and cell culture

Gyp, dimethyl sulfoxide (DMSO), potassium phosphates, Propidium iodide (PI), ribonuclease-A, Tris-HCl, Trypan blue and Triton X-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). TE buffer was purchased from Merck Co. (Darmstadt, Germany). 2,7-Dichlorodihydrofluorescein diacetate, DiOC6 and Indo 1/AM were obtained from Calbiochem (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, fetal bovine serum (FBS) and penicillin–streptomycin, trypsin-EDTA were

Effects of Gyp on morphology and viability of SCC-4 cells

After the SCC-4 cells were exposed to different concentrations of Gyp and different time-periods, cells were photographed, before being collected for propidium iodine staining for viability analysis. It can be seen in Figure 1A that the cells were morphologically-altered by Gyp treatment. Fig. 1B shows that there were fewer viable cells as time and concentration increased.

Gyp effects on cell cycle arrest and apoptosis in SCC-4 cells

Cell cycle and apoptosis analysis indicated that, during 48-h Gyp treatment, there was an increase in the percentage of

Discussion

Our earlier studies showed that Gyp inhibited N-acetyltransferase activity and gene expression in human cervical cancer Ca Ski cells.15 Recently, we also found that Gyp induced apoptosis in human colon cancer which was the mitochondria-dependent and involved caspase-3 activation.14 There is no report to show Gyp affect oral cancer. Therefore, in the present study, Gyp induced G0/G1 arrest and apoptosis in human tongue cancer SCC-4 cells, thus providing a useful model system to characterize the

Conflict of interest statement

None declared.

Acknowledgement

This work was supported by Grants CMU95-327 and CMU95-330 from the China Medical University of Taiwan.

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