Ethanol elicits inhibitory effect on the growth and proliferation of tongue carcinoma cells by inducing cell cycle arrest

Abstract

Cellular effects of ethanol in YD-15 tongue carcinoma cells were assessed by MTT assay, caspase activity assay, Western blotting and flow cytometry. Ethanol inhibited the growth and proliferation of YD-15 cells in a dose- and time-dependent manner in an MTT assay. The effects of ethanol on cell cycle control at low percent range of ethanol concentration (0 to 1.5%), the condition not inducing YD-15 cell death, was investigated after exposing cells to alcohol for a certain period of time. Western blotting on the expression of cell cycle inhibitors showed that p21 and p27 was up-regulated as ethanol concentration increases from 0 to 1.5% whilst the cell cycle regulators, cdk1, cdk2, and cdk4 as well as Cyclin A, Cyclin B1 and Cyclin E1, were gradually down-regulated. Flow cytometric analysis of cell cycle distribution revealed that YD-15 cells exposed to 1.5% ethanol for 24 h was mainly arrested at G2/M phase. However, ethanol induced apoptosis in YD-15 cells exposed to 2.5% or higher percent of ethanol. The cleaved PARP, a marker of caspase-3 mediated apoptosis, and the activation of caspase-3 and -7 were detected by caspase activity assay or Western blotting. Our results suggest that ethanol elicits inhibitory effect on the growth and proliferation of YD-15 tongue carcinoma cells by mediating cell cycle arrest at G2/M at low concentration range and ultimately induces apoptosis under the condition of high concentration.

Keywords: Cell cycle arrest; Cell growth; Ethanol; Tongue carcinoma cell.

Fig. 1

Ethanol effects on the growth of cancer cells. Different types of cancer cells were treated with various concentrations of ethanol for 24 h and cell growth was evaluated by MTT assay. The growth of YD-15 cells showed the highest sensitivity to ethanol among the investigated cell types. Vertical bars indicate means and standard errors (n=3). ^*p<0.01, versus FaDu; ^**p<0.05, versus HeLa cells.

Fig. 2

Ethanol dependence of cancer cell proliferation. Cell proliferation of two types of oral cancer cells (YD-15 cells and FaDu cells) were determined by an MTT assay. Cells were treated for a given incubation periods at a fixed ethanol concentration prior to the assay. (A) YD-15 cells. (B) FaDu cells. Vertical bars indicate means and standard errors (n=3).

Fig. 3

The expression of cell cycle inhibitors, p21 and p27, increased after ethanol treatment of YD-15 cells at different concentrations for 24 h.

Fig. 4

Ethanol effects on the expression of cell cycle regulators. YD-15 cells were exposed with various ethanol concentrations for 24 h. (A) Cyclin expression. (B) Cyclin dependent kinase expression.

Fig. 5

Cell cycle profiles by flow cytometry analysis. YD-15 cells were treated with ethanol in culture medium for 24 h. (A) Without ethanol treatment. (B) With 1.5% ethanol treatment.

Fig. 6

The expression of apoptosis associated proteins in YD-15 cells. Bcl-2, Bad, Bax, inactivated PARP (85 kDa) and activated caspase-7 (20 kDa) were detected by Western blotting after treating YD-15 cells with ethanol for 24 h. (A) Expression of Bcl-2, Bax, Bad and PARP after exposing cells in the range of 0.25% to 1.5% ethanol. (B) PARP and caspase-7 expression after treating cells with 2.5% ethanol. (C) Caspase activity in arbitrary units. Activated caspase-3 and -7 were detected by using Caspase-Glo® 3/7 Assay kit (Promega, Madison, WI). Vertical bars indicate means and standard errors (n=3).