Butyrate suppresses mRNA increase of osteopontin and cyclooxygenase-2 in human colon tumor tissue

Abstract

The short chain fatty acid (SCFA) butyrate, a product of fermentation of dietary fiber in the human colon, is found to exert multiple regulatory processes in colon carcinogenesis. The aim of this study was to find out whether butyrate affects the tumor-promoting genes osteopontin (OPN) and cyclooxygenase (COX)-2, their respective proteins and/or their functional activity in matched normal, adenoma and tumor colon tissues obtained from 20 individuals at colon cancer surgery. Quantitative real-time polymerase chain reaction experiments showed increased levels of OPN and COX-2 messenger RNA in tumor tissues when compared with the adjacent normal samples (P < 0.001). The addition of butyrate reduced OPN and COX-2 mRNA expression in all tissue types compared with the related medium controls (tumor: P < 0.05). In tumor samples, a downregulation of up to median 35% (COX-2) and 50% (OPN) was observed, respectively. Thereby, tumors with lower levels of OPN basal expression were more sensitive to inhibition and vice versa for COX-2 in normal tissue. At the protein and enzyme level, which were determined by using western blot and enzyme immunometric assays, the impact of the SCFA was not clearly visible anymore. The active proteins of OPN and COX-2 (determined by prostaglandin E(2)) were found to correlate with their respective mRNA expression only in 50-63% of analyzed donors. For the first time, our data reveal new insights into the chemoprotective potential of butyrate by showing the suppression of OPN and COX-2 mRNA in primary human colon tissue with the strongest effects observed in tumors.

Impact of butyrate on PKM2 and HSP90β expression in human colon tissues of different transformation stages: a comparison of gene and protein data

“…Normal tissues showed no signs of malignancy and inflammation and adenoma samples were proven to be benign. The tissue samples were stored in Hank's balanced salt solution (HBSS; 8.0 g/l NaCl; 0.4 g/l KCl; 0.06 g/l Na 2 HPO 4 9 2 H 2 O; 0.06 g/l K 2 HPO 4 ; 1 g/l glucose; 0.35 g/l NaHCO 3 ; 4.8 g/l HEPES; pH 7.2) on ice and prepared immediately as previously reported (Jahns et al 2011). The resulting tissue strips of normal, adenoma, and tumor samples were either frozen in liquid nitrogen alone or submerged in RNA later (Qiagen, Hilden, Germany) and stored at -80°C until cytosol or RNA was being extracted.…”

“…Treatment with the same batch of an intestinally relevant dose of butyrate (10 mM) (Hamer et al 2008) for 12 h was performed in parallel, subsequently (Jahns et al 2011). Primary cell culture medium (solvent for butyrate) was used as a control in the experiments and consisted of minimal essential medium with Earle's salts enriched with 20% fetal calf serum, 2 mM glutamine, 100 lg/ml gentamycin, 2.5 lg/ml fungizone, 10 ng/ml epidermal growth factor, 5 lg/ml insulin, 5 lg/ml transferrin, and 5 ng/ml sodium selenite according to Rogler et al (1998).…”

“…Isolation of RNA and reverse transcription into complementary DNA (cDNA) RNA isolation and cDNA synthesis were performed as previously described (Jahns et al 2011). In short, total RNA was isolated from the homogenized tissue samples by using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol followed by spectrophotometrically quantification with the NanoDrop Ò ND-1000 (NanoDrop Technologies, Wilmington, DE).…”

“…Quantitative PCR conditions were already described by Jahns et al (2011). Briefly, 2 ll cDNA prepared from different starting concentrations of total RNA were amplified in three steps by using iQ SYBR Green Supermix (Bio-Rad, Munich, Germany) and 10 pmol gene-specific primers: HSP90b forward, 5 0 -CGTTGCTCACTATTACG TATAATCCT-3 0 and reverse, 5…”

“…Subsequently, the membrane was blocked with 5% nonfat dried milk powder (1 h; AppliChem, Darmstadt, Germany) and incubated with the following primary and secondary antibodies: HSP90b (Zymed Laboratories, San Francisco, CA), PKM2 (Cell Signaling Technology, Danvers, MA), b-actin (Sigma-Aldrich, Steinheim, Germany), Polyclonal Rabbit Anti-Mouse IgG/HRP (Dako, Hamburg, Germany), and Polyclonal Goat Anti-Rabbit IgG/HRP (Dako). Detection and evaluation of protein bands were performed as previously described (Jahns et al 2011).…”