The Effects of Liquor Spirits on RNA Pol III Genes and Cell Growth of Human Cancer Lines

Alcohol consumption is a major health issue and associated with human cancers, such as liver and breast cancers. Alcohol was classed as carcinogen to human by IARC. We have performed in vivo and in vitro studies which demonstrate that diluted ethanol promotes cell proliferation and transformation and tumor formation. Consumption of liquor spirits (white wines) is a popular behavior. However, it is unclear whether liquor spirits affect cellular phenotypes of human cancers. At present study, we used diluted ethanol and liquor spirits (Sample #1 and Sample #2) to determine the changes in RNA polymerase III-dependent gene (Pol III gene) transcription, cell growth and colony formation in the different human cancer lines. The results indicate that low concentration of ethanol increases RNA Pol III gene transcription and rate of cell growth. However, both liquor spirits (Sample #1 and Sample #2) inhibit the activity of RNA Pol III genes and repress cell proliferation of the cancer lines, compared to diluted ethanol. The liquor spirits reduce the rate of colony formation of human breast cancer cells and esophageal carcinoma cells. The inhibitions of the liquor spirits to RNA Pol III genes, cell growth and colony formation are in a dose-dependent manner. These new findings suggest that the liquor spirits contain some active components to repress Pol III gene transcription and cell growth caused by ethanol in different human cancer cells.


Introduction
Numerous studies have indicated that alcohol intake is associated with human cancers in different organs, such as breast, liver, stomach, pancreas, oral cavity, pharynx, esophagus, larynx, colon and ovary [1]- [8]. Alcohol has been classed as a carcinogen to human by international agency for research on cancer (IARC) [9] [10]. This implies that there may exist an underlying mechanism, by which alcohol promotes human cancer development. However, the details of the mechanism remain to be elucidated. Nucleolar hypertrophy is a consistent cytological feature of cancer cells, where RNA polymerase III-dependent genes (Pol III genes) are transcribed. Upregulation of Pol III genes is tightly linked to cell proliferation, cell transformation and tumor development [11] [12] [13]. Our studies in vivo and in vitro have demonstrated that alcohol treatment enhances transcription of Pol III genes to promote alteration of these cellular phenotypes [13] [14] [15]. This suggests that upregulation of Pol III genes caused by ethanol plays an important role in cancer development.
The fibrosis and cirrhosis of liver are the key processes during HCC development. Ethanol exposure increases cellular production of reactive oxygen species, causing cellular stress and resulting in liver injury and alcoholic liver disease (ALD) [26]. Alcohol is known to promote liver and mammary tumorigenesis [27] [28] [29] [30]. Our studies have demonstrated that alcohol activates JNK1 to upregulate Pol III gene transcription [14] [15]. Activation of JNK1 increases the rate of cell proliferation and enhances cell transformation and colony formation [12] [13]. Therefore, alcohol can be used as an agent to determine changes in these cellular phenotypes.
There is a long history of liquor spirits (white wine) consumption in China and the world. The liquor spirits of China were produced by using rice, corn, sorghum, and other grains by fermenting and distilling processes. In terms of difference of producing procedures, the liquor spirits of China were divided two types: Nong-Xiang liquors (Sample #1) and Jiang-Xiang liquors (Sample #2).
Both of them contain slightly over 50% ethanol (v/v). Epidemiological study indicated that the workers, who tested Jiang-Xiang liquor to check its quality during the course of production, were don't found hepatic fibrosis, cirrhosis and HCC in the special crowd, compared to a group with other beverage consumption [31]. Our study indicated that alcohol-feeding of HCV NS5A transgenic mice with 3.5% ethanol induces liver steatosis and inflammation to promote HCC development [14] [30]. DEN (Diethylnitrosamine), a potent chemical hepatocarcinogen, has widely been used to induce HCC in rodents. DEN adminis-

RNA Isolation and RT-qPCR
Total RNA was isolated from human breast cancer cell line (MCF-7) and eso- in DNase-free water and real time qPCR (RT-qPCR) were performed with specific primers as described previously [12] [15] and PCR reagent kits (Bio-Rad     (Figure 1(a1) and Figure 1(b1)) or 5S rRNA (Figure 1(a2) and Figure 1(b2)) genes in human breast cancer cell line (MCF-7) (Figure 1(a)) and esophageal carcinoma cell line (KYSE-510) (Figure 1(b)). The results are consistent with our early studies [14] [15]. In contrast, the liquor spirits, both Sample #1 and Sample #2, significantly inhibit the gene activity at same concentration of actual ethanol (p < 0.01). Next, we determined the effect of liquor spirits on cell growth. The breast cancer MCF-7 cells grow to almost full confluence at diluted ethanol-treated cells from 12.5 mM to 200 mM of ethanol (Figure 2(a)). At lower concentration of ethanol    As inhibiting Pol III gene transcription is able to repress cell transformation [11] [12] [33], ethanol treatment promotes the transformation of normal cells and increases colony formation of cancer cells [11] [12]. Here, our data indicate that liquor spirits inhibit Pol III gene transcription (Figure 1(a)). Therefore, we

Declaration of Interest
The authors declare that they have no conflict of interest.