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.
Ethanol Liquor Spirits Cancer Cells Pol III Genes Cell Growth Colony Formation1. 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.
Breast cancer is the major malignant neoplasms of women in the United States. Studies indicate that alcohol consumption is an established risk factor of breast cancer [16] - [21] . The relative increase in risk ranges from 5% - 10% (~1 drink/10 gram/day) to 40% - 50% (~3 drinks/day) [22] [23] . Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, eventually creates cirrhosis, increasing the risk of hepatocellular carcinoma (HCC) [14] [24] [25] . 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 administration caused HCC in 100% of male mice [32] . Our studies have demonstrated that DEN increases Pol III gene transcription to enhance cell proliferation and cell transformation of mouse liver cells [33] . Male C57BL/6J mice were injected with DEN and then treated with Jiang-Xiang Liquor, compared to diluted ethanol at same concentration [34] . The incidence of HCC in Jiang-Xiang Liquor-fed group of mice is significant lower than diluted ethanol-fed group [34] . However, DEN is not really associated with human. To investigate whether the liquor spirits are indeed able to inhibit cell growth of human cancers, we have utilized two liquor spirits of China, a Nong-Xiang liquor (Sample #1) and a Jiang-Xiang liquor (Sample #2), to determine whether the two liquor spirits affect RNA Pol III gene transcription and cellular phenotypes of different human cancer lines. The results reveal that both liquor spirits (Sample #1 and Sample #2) inhibit Pol III gene activity and decrease the rates of cell proliferation of these cancer lines, compared to diluted ethanol treatment. The two liquor spirits also inhibit colony formation of human breast cancer and esophageal carcinoma lines. These studies suggest that the liquor spirits may contain some bioactive components, which are indeed of capacity of repressing ethanol-induced Pol III gene transcription and decreasing ethanol-enhanced the rates of cell growth and colony formation in different human cancer lines.
2. Materials and Methods2.1. Cell Lines and Reagents
Human breast adenocarcinoma cell line MCF-7 (HTB-22), human colon carcinoma cell line RKO (CRL-2577), human colorectal carcinoma cell line HCT-116 (CRL-247), human colorectal adenocarcinoma cell line SW-480 (CCL-228) were from ATCC. Human esophageal squamous cell carcinoma line, KYSE-510 (ACC 374) was from DSMZ. These cell growth media were from Life Technologies Inc.
Liquor spirits (white wines) of China, a Nong-Xiang liquor (52% ethanol, v/v) as Sample #1 and a Jiang-Xiang liquor (53% ethanol, v/v) as Sample #2. Both liquor spirits were gifts from Dr. Zhenghong Xu (Jiangnan University of China). Absolute ethanol (200 proof) was from Sigma-Aldrich. The cells were treated with ethanol or liquor spirits (Sample #1 or Sample #2) at corresponding actual ethanol concentration (mM/L).
2.2.RNA Isolation and RT-qPCR
Total RNA was isolated from human breast cancer cell line (MCF-7) and esophageal carcinoma cell line (KYSE-510) treated with liquor spirits or diluted ethanol at same concentration of actual ethanol as indicated in figures using single step extraction method TRIzol reagent (Invitrogen). RNA samples were quantified and reverse-transcribed in a 20 µl reaction containing 1 × RT (reverse transcription) buffer. After first-strand cDNA synthesis, the cDNAs were diluted 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 Biotech) in the ABI prism 7700 Sequence Detection System. Precursor of tRNALeu and 5S rRNA transcripts were measured by RT-qPCT [13] [14] .
2.3. Cell Proliferation
Approximately 2 × 103 cells per ml of human cancer cells (MCF-7, KYSE-510, RKO, HCT-116, SW-480) were seeded in six-well plates in triplicate. The cells were treated with liquor spirits or diluted ethanol at same concentration of actual ethanol as indicated in figures. The confluences of the cells in 6 days were about 80% ~ 90%. The cell morphology was analyzed by microscopy using a Nikon Eclipse TE300 and Metamorph Program (Cell and Tissue Imaging Core of University of Southern California Research Center for Liver Diseases, P30 DK048522). Cells were assayed for viability and counted using a Coulter counter [12] [13] .
2.4. Cell Anchorage-Independent Growth
MCF-7 cells and KYSE-510 cells (1 × 104 cells/well in 6 well plate) were suspended in 0.35% (w/v) agar in 10% FBS/DMEM/F12 or RIPM1640 media with specified concentrations of diluted ethanol or liquor spirit (Sample #1 and Sample #2) as indicated in Figure 6. Cells were fed fresh complete media with diluted ethanol, liquor spirits Sample #1 or Sample #2 at same concentration of actual ethanol twice weekly. Colonies were counted 2 - 3 weeks or longer after plating as described previously [11] [12] .
3. Results and Discussion
Our previous studies have reported that diluted ethanol at 25 ~ 50 mM (the original ethanol as 200 Proof), increases Pol III gene transcription in cell culture models [14] [15] . We have demonstrated that enhancement of Pol III gene transcription promotes cell proliferation and cell transformation [12] [13] . The Pol III gene transcription was increased in liver tumor tissues of ethanol-fed mice, compared to non-tumor tissues [14] . Repression of Pol III gene transcription results in decreasing the rates of cell proliferation and colony formation [11] [12] [13] . Therefore, reduction of the cellular phenotypic changes stands for repression of Pol III gene transcription. Study indicated that intake of a Chinese liquor spirit repressed DEN-induced liver tumor formation of mice, compared to the ethanol-fed group at same doses [34] . An epidemiological study that workers consumed the Chinese liquor spirit longer than ten years indicated that there not was hepatic fibrosis or cirrhosis in the special group, compared to a group with other beverage sources [31] . Fibrosis and cirrhosis of liver are key processes during HCC development. The liquor spirits of China are distilled liquors and made by grains through fermenting and distilling processes. It implies that the liquor spirits may have potential to repress liver cancer development.
To explore the roles of liquor spirits (Sample #1 and Sample #2) in alterations of different human cancer cells, we have determined Pol III gene transcription, cell proliferation and colony formation. The results show that diluted ethanol at 25 mM increases Pol III gene transcription, either tRNALeu (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
(from 12.5 mM to 25 mM ethanol), accumulation of the cells was increased (Figure 2(a) and Figure 2(d)). Then, the numbers of cells were then slightly decreased with enhancing ethanol doses (Figure 2(a) and Figure 2(d) left). While the liquor spirits, both Sample 1# and Sample #2, reduce the rate of MCF-7 cell growth (Figures 2(b)-(d)). At the concentration of 100 mM ethanol of Sample #2 almost completely inhibits the MCF-7 cell growth (Figures 2(b)-(d)), whereas Sample #1 also dramatically represses the cells (Figure 2, middle and right). These studies show that the liquor spirits are indeed able to inhibit the activity of Pol III genes and repress cell growth of human breast cancer.
We have also determined the effects of liquor spirits on the cell growth of human esophageal carcinoma line, KYSE-510. The accumulation of the KYSE-510 cells is increased by ethanol at 12.5 mM and 25 mM, whereas higher dose of the diluted ethanol not further increase the numbers of the cells (Figure 3(a) and Figure 3(d) left). At low dose of 12.5 mM ethanol, both liquor spirits slightly decrease the cell growth. However, the liquor spirits dramatically reduce
the rate of KYSE-510 cell proliferation at 100 mM and 200 mM ethanol (Figure 3(d) middle and right). The results indicate that this cell line is more sensitive to liquor spirits, Sample #1 than Sample #2 (Figures 3(b)-(d)) at the high doses of actual ethanol. To further explore the effects of the liquor spirits on cancer cell growth, we have used more human cancer lines, such as HCT-116, SW-480 and RKO. The results have shown that diluted ethanol at low doses (12.5 mM to 25 mM) increase HCT-116 and SW-480 the rate of cell growth (Figure 4(a) and Figure 4(b)). While the liquor spirits, Sample #1 and Sample #2, from low doses to high one significantly reduces the acuminated numbers of the human colon carcinoma cells of HCT-116, SW-480 and RKO lines (Figure 4). In order to confirming the effects of liquor spirits on cancer cells, we have additionally tested the effects of the liquor spirits on the tumor stem cells of mouse liver (TSCML). The two liquor spirits reduce the rate of cell proliferation of TSCML (Figure 5). But, the repressing cell growth of TSCML by liquor spirit, Sample #2 is more significant than Sample #1 (Figure 5). Together, the above studies have shown that ethanol increases the rate of cell proliferation of the cancer cell lines
at lower concentration. Although the accumulation of the KYSE-510 cells is decreased at high dose of diluted ethanol, the higher dose of ethanol induces cellular apoptosis. While liquor spirits, both Samples #1 and Sample #2, display the inhibition of cell growth of different cancer lines from low to high concentration. The inhibiting roles of liquor spirits in Pol III gene transcription and cell growth are in a dose-dependent manner.
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 further determine the effect of liquor spirits on colony formation of the human cancer cells. We seeded the human breast cancer line, MCF-7 cells (Figure 6 top) and human esophageal carcinoma line, KYSE-510 cells (Figure 6 bottom) in soft agar and treated the cells with the liquor spirits, respectively. The results indicate that the liquor spirits, either Sample #1 (Figure 6 left) or Sample #2
(Figure 6 right) inhibit colony formation of the human cancer cells (Figure 6).
In summary, the present studies indicate that the liquor spirits display the effects of repression on Pol III gene transcription, cell proliferation and colony formation in different human cancer lines. It implies that liquor spirits may contain some bioactive compounds which inhibit ethanol-induced RNA Pol III gene activity and ethanol-caused cancer cellular phenotypic alteration. This is the first report on the liquor spirits repressing cell growth of different human cancer lines. It suggests that identifying the bioactive compounds of the liquor spirits and enhancing the ratio of the compounds during producing processes will benefit drinkers to prevent cancer development.
Acknowledgements
This work was supported by CNJ14C007 to Y.Y.
Declaration of Interest
The authors declare that they have no conflict of interest.
Author Contributions
YY and SZ involved in conception and design and in analysis and interpretation of data. GS, and SZ involved in data acquisition and in writing and reviewing of the manuscript. YY, JL, SC, YZ and ZH involved in development of methodology. SC, JL, YZ, YY provided administrative, technical, or material support. SZ and GS supervised the study. All authors read and approved the final manuscript.
Cite this paper
Yi, Y.F., Lei, J.X., Shi, G.G., Chen, S.L., Zhang, Y.M., Hong, Z.F., He, Z.M. and Zhong, S.P. (2018) The Effects of Liquor Spirits on RNA Pol III Genes and Cell Growth of Human Cancer Lines. Food and Nutrition Sciences, 9, 208-220. https://doi.org/10.4236/fns.2018.93016
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