Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP)

Abstract

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspasecascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently upregulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrateinduced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 ± 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspasecascade and the mitochondrial bcl-pathway.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

“…The morphological features of apoptosis such as nuclear condensation and fragmentation of genomic DNA were clearly indicative in BuA-treated EAT cells. Caspase proteases play a crucial role in inducing apoptosis, and BuA has been shown to induce the activation of caspases during apoptosis [20,21]. Our studies demonstrate that Ac-DEVD-CHO, a specific caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, suggesting that BuAinduced apoptosis in EAT cells is mediated via the caspase-3 pathway.…”

“…Our studies demonstrate that Ac-DEVD-CHO, a specific caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, suggesting that BuAinduced apoptosis in EAT cells is mediated via the caspase-3 pathway. Activation of caspase-3 has been shown to act on downstream targets such as the DNA-repair enzyme poly (ADP-ribose) polymerase (PARP) or DNA fragmentation factor (DEF), leading to the breaking of DNA strands into 180 bp pieces [20,41]. It is evident from our results that BuA can activate CAD, which is associated with its inhibitor ICAD in the cytosol [42].…”

“…Several studies have reported that BuA induces apoptosis in a variety of cell types [20][21][22][23][24][25]. Hence, an attempt was made to identify whether the BuA-induced antiproliferative effect on EAT cells was due to induction of apoptosis.…”

“…It is well recognized that various caspases play a crucial role in apoptosis. We examined whether or not BuA has any effect on the activation of caspase-3, a cysteine protease responsible for morphological changes and DNA degradation in apoptotic cells [20,21]. Preincubation of EAT cells with caspase-3 inhibitor completely inhibited BuA induced nuclear condensation (data not shown) and DNA degradation (Fig.…”

“…The inhibitory role of BuA against tumor growth is mediated through a direct effect on tumor cells that results in cell cycle arrest, differentiation, or apoptosis [16][17][18][19]. BuA-induced apoptosis has been shown to involve activated caspase-3 in several cell lines [20,21]. In addition, BuA has also been shown to activate p21, a cyclin-dependent kinase inhibitor, independent of p53 activation to induce cell death [22][23][24][25].…”