Efficacy of Butyrate to Inhibit Colonic Cancer Cell Growth Is Cell Type-Specific and Apoptosis-Dependent.

Increasing dietary fiber consumption is linked to lower colon cancer incidence, and this anticancer effect is tied to elevated levels of short-chain fatty acids (e.g., butyrate) because of the fermentation of fiber by colonic bacteria. While butyrate inhibits cancer cell proliferation, the impact on cancer cell type remains largely unknown. To test the hypothesis that butyrate displays different inhibitory potentials due to cancer cell type, we determined half-maximal inhibitory concentrations (IC50) of butyrate in HCT116, HT-29, and Caco-2 human colon cancer cell proliferation at 24, 48, and 72 h. The IC50 (mM) butyrate concentrations of HCT116, HT-29, and Caco-2 cells were [24 h, 1.14; 48 h, 0.83; 72 h, 0.86], [24 h, N/D; 48 h, 2.42; 72 h, 2.15], and [24 h, N/D; 48 h, N/D; 72 h, 2.15], respectively. At the molecular level, phosphorylated ERK1/2 and c-Myc survival signals were decreased by (>30%) in HCT116, HT-29, and Caco-2 cells treated with 4 mM butyrate. Conversely, butyrate displayed a stronger potential (>1-fold) for inducing apoptosis and nuclear p21 tumor suppressor in HCT116 cells compared to HT-29 and Caco-2 cells. Moreover, survival analysis demonstrated that a cohort with high p21 gene expression in their colon tissue significantly increased survival time compared to a low-p21-expression cohort of colon cancer patients. Collectively, the inhibitory efficacy of butyrate is cell type-specific and apoptosis-dependent.

Methylselenol, a selenium metabolite, plays common and different roles in cancerous colon HCT116 cell and noncancerous NCM460 colon cell proliferation.

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action.

Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75-300 micromol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

The selenium metabolite methylselenol inhibits the migration and invasion potential of HT1080 tumor cells.

There is increasing evidence for the efficacy of certain forms of selenium as cancer-chemopreventive compounds. Methylselenol has been hypothesized to be a critical selenium metabolite for anticancer activity in vivo. To determine whether tumor cell migration, invasion, and cell cycle characteristics are inhibited by methylselenol, we exposed HT1080 cells to methylselenol. Methylselenol was generated with seleno-L-methionine (a substrate for methioninase). Submicromolar methylselenol exposure led to an increase in the G1 and G2 fractions with a concomitant drop in the S-phase, indicating slower cell growth. Furthermore, methylselenol inhibited the migration and invasion rate of the tumor cells by up to 53 and 76%, respectively, when compared with the control tumor cells. Although all cells had increased matrix metalloproteinase (MMP) enzyme activities of pro-MMP-2 and pro-MMP-9, the active form of MMP-2 was decreased in HT1080 cells cultured with methylselenol. In addition, methylselenol increased the protein levels of antimetastasic tissue inhibitor metalloproteinase (TIMP)-1 and TIMP-2. Collectively, these results demonstrate that submicromolar concentrations of methylselenol increase both prometastasis MMP-2 and MMP-9 and antimetastasis TIMP-1 and TIMP-2 expression. The apparent net effect of these changes is the inhibition of pro-MMP-2 activation and carcinogenic potential or activity.

Prolonged butyrate treatment inhibits the migration and invasion potential of HT1080 tumor cells.

Butyrate, a normal constituent of the colonic luminal contents, is produced by the bacterial fermentation of dietary fibers and resistant starches. It has been hypothesized that butyrate may inhibit the invasion of tumor cells. The purpose of the present study was to investigate the effects of butyrate treatment on the growth, migration, and invasion characteristics of tumor HT1080 cells. HT1080 cells cultured in the presence of 0.5 and 1 mmol/L butyrate for 14 d exhibited an increase in the G(1) and G(2) fractions with a concomitant drop in the S-phase, thus showing slower cell growth. Interestingly, 0.5 and 1 mmol/L butyrate inhibited the migration and invasion rate of the tumor cells compared with the untreated (control) cells. The protein and mRNA levels of the tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were significantly increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. Enzymatic activities and the mRNA level of the latent forms of matrix metalloproteinase (MMP), pro-MMP-2 and pro-MMP-9, were also increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. In contrast, the active form of MMP-2 was detectable by zymographic analysis in control but not butyrate-conditioned media. Collectively, these results demonstrate that prolonged and low-dose butyrate treatment increases both prometastasis MMP-2, -9 and antimetastasis TIMP-1, -2 expression, and the net effect of these increases is the inhibition of pro-MMP-2 activation and of tumor cell migration/invasion potential.

Fatty acids alter monolayer integrity, paracellular transport, and iron uptake and transport in Caco-2 cells.

The Caco-2 cell line was used as a model to determine if the type of fatty acid, unsaturated versus saturated, differentially altered the uptake and transport of iron in the human intestine and if the changes were the result of alterations in monolayer integrity and paracellular transport. Cells were cultured in either a lower-iron or normal-iron medium and incubated with a bovine serum albumin control, linoleate, oleate, palmatate, or stearate. Oleate, palmatate, and stearate enhanced (p < 0.05) iron uptake in cells grown in lower iron. The fatty acid effect on iron uptake by cells grown in normal iron was not as pronounced. Iron transport was not affected (p > 0.05) by an interaction between the type of medium (iron concentration) and the type of fatty acid. Iron transport was enhanced (p < 0.05) with palmatate and stearate. Various indicators of monolayer integrity and paracellular transport were also affected by the fatty acids, thus impacting iron uptake and transport. These results indicate that oleate, palmatate, and stearic can enhance iron uptake and transport; however, this enhancement may be the result of alterations in the integrity of the intestine.