Milk lactoperoxidase decreases ID1 and ID3 expression in human oral squamous cell carcinoma cell lines

Abstract

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

Figure 1

STRING analysis of milk-regulated genes in HSC2 cells. HSC2 cells when exposed to 5% aqueous fraction of pasteurized human milk express genes that were revealed by whole-genome gene array that can be clustered by STRING analysis.

Figure 2

Decreased ID1, ID3 and DLX2 expression in HSC2 exposed to milk. HSC2 oral squamous cell carcinoma cells were exposed to 5% of aqueous fractions of pasteurized human milk (A), cow’s milk (B) and infant formula (C) for 24 hours, before expression analysis of the target genes ID1, ID3 and DLX2 were performed. Data indicate the x-fold decrease normalized to unstimulated control cells. The experiments performed at least three times.

Figure 3

Decreased ID1, ID3 and DLX2 expression in TR146 exposed to milk. TR146 oral squamous cell carcinoma cells were exposed to 5% of aqueous fractions of pasteurized human milk (A), cow’s milk (B), and infant formula (C) for 24 hours, before expression analysis of the target genes ID1, ID3 and DLX2 were performed. Data indicate the x-fold decrease normalized to unstimulated control cells. The experiments performed at least three times.

Figure 4

Western blot of ID1, ID3 and DLX2 in HSC2 exposed to milk. HSC2 oral squamous cell carcinoma cells were exposed to 5% of aqueous fractions of pasteurized human milk (MM), cow’s milk (CM), and infant formula (IF) for 72 hours. (A) Western blot analysis of ID1, ID3 and DLX2 was performed. (B) Data indicate the relative changes normalized to actin and unstimulated control cells.

Figure 5

TGF-β receptor and MAPK inhibitors in HSC2 cells exposed to human milk. HSC2 oral squamous cell carcinoma cells were exposed to 5% of aqueous fractions of pasteurized human milk in the presence and absence of SB431542, a TGF-β receptor I kinase inhibitor, and inhibitors against ERK, p38 and JNK signaling, U0126, SB203580, SP600125, respectively. Expression analysis of ID1 (A), ID3 (B) and DLX2 (C) was performed. Data indicate the x-fold decrease compared to unstimulated control cells. (D) HSC2 oral squamous cell carcinoma cells were exposed to 5% of aqueous fractions of pasteurized human milk, inflammation provoked by IL1β and TNFα Western blot analysis shows increased phosphorylation signals of particular ERK and to a lesser extent p38 and JNK by exposing HSC2 cells to milk for 30 minutes. (E) Data indicate the relative changes normalized to unphosphorylated MAPK and unstimulated control cells. The experiments performed at least three times.