Role of betaine in inhibiting the induction of RNA Pol III gene transcription and cell growth caused by alcohol

Abstract

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.

Keywords: Alcohol; Betaine; Brf1; Cell growth; Colony formation; Pol III genes.

Fig 1.

Role of betaine in inhibition of MCF-7 cell growth. 3000 MCF-7 cells were cultured with 3 ml medium per well in 6-well plates. Dose curve: ethanol (50 mM) and different amounts of betaine were added into the medium at the 0 hour, the 48^th hour and the 96^th hour, respectively. The pictures were taken under a microscope at the 6^th day. Original magnification X 40.

Fig. 2.

Betaine represses cell growth of human breast cancer. (A) Time curve, the ethanol (50 mM) and betaine (40 mM) were added in the medium, and the total cell numbers were counted from the first day to the sixth day. (B) The cells were treated with or without ethanol and different doses of betaine. The total cell numbers were counted after 6 days. * P<0.05 vs control (Ethanol=0, Betaine=0).

Fig 3.

The role of betaine in MCF-7 cell growth by MTT assay. Approximately 300 MCF-7 cells were cultured with 300 μl medium per well in 96-well plates. (A) Dose curve. The ethanol (50 mM) and different concentrations of betaine were added into the medium at the 0 hour, the 48^th hour and the 96^th hour, respectively. OD value was detected after treated the cells for 6 days. (B) Time curve. The concentration of ethanol and betaine were 50 mM and 40 mM, respectively. OD value was detected every day after plating.

Fig 4.

Colony formation assay. 1000 MCF-7 cells were planted in the upper agar in 6-well plates. Ethanol (50 mM) and betaine (40 mM) were added at the 1^st and 4^th, respectively. The result indicates that betaine is able to reduce the rate of MCF-7 cell colony formation caused by ethanol. *P < 0.01; **P < 0.01.

Fig 5.

Betaine represses Brf1 expression. MCF-7 cells were pre-treated with betaine for 2 hours and then treated with ethanol for another 1 hour. (A) Immunoblot analysis was performed using protein lysates derived from these cells to detect the Brf1 protein level. Actin was used as a loading control. A represent immunoblot (A, Left panel). A densitometric analysis are revealed and the fold changes in Brf1 level were normalized by actin (A, Right panel). (B) Brf1 mRNA level. MCF-7 cells were treated with alcohol (50mM) and betaine as indicated above, to extract total RNA. mRNA levels of Brf1 were measured by RT-PCR. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent Mean ± SE of at least three independent determinations. These results indicate that betaine reduces the induction of Brf1 expression caused by alcohol.

Fig 6.

Betaine mediates Pol III gene transcription. MCF-7 cells were treated as indicated above. The amounts of pre-tRNA^Leu (A) and 5S rRNA (B) were measured by RT-qPCR. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent Mean ± SE of at least three independent determinations.

Fig 7.

Schematic illustration of the role of betaine in Brf1 expression and Pol III gene transcription. Alcohol increases Brf1 expression to elevate Pol III gene transcription and to promote cell proliferation and transformation, eventually resulting in breast cancer development. In contrast, betaine inhibits the induction of these genes caused by alcohol to decrease Brf1 expression and Pol III gene transcription, leading to repression of breast cancer cell growth and tumor development.