Dissociation of staurosporine-induced apoptosis from G2-M arrest in SW620 human colonic carcinoma cells: initiation of the apoptotic cascade is associated with elevation of the mitochondrial membrane potential (deltapsim).

We have identified an alternative apoptotic cascade induced in SW620 human colonic carcinoma cells by the protein kinase antagonist staurosporine (stsp). Consistent with its effect in other colonic epithelial cells, stsp induced G2-M arrest and apoptosis of SW620 cells. However, despite the paradigm that growth arrest triggers apoptotic cascades, apoptosis was detected before G2-M arrest. Reports have linked dissipation of the mitochondrial membrane potential (deltapsim) to the initiation of apoptosis and have linked elevation of the deltapsim to the escape from apoptosis However, neither apoptosis nor cell cycle arrest were altered by the collapse of the deltapsim, and increased deltapsim enhanced the initiation of apoptosis but blocked G2-M arrest. Although reactive oxygen species (ROS) have been implicated in some colonic epithelial cell and stsp-induced cascades, neither antioxidants nor the inhibition of RNA or protein synthesis altered apoptosis of SW620 cells. Finally, cytosolic cytochrome c has been linked to activation of caspase-3 and dissipation of the deltapsim. However, caspase-3 activation preceded the accumulation of cytochrome c in the cytosol and was accompanied by transient elevations in both the deltapsim and mitochondria-associated cytochrome c. Therefore, we have identified a distinct apoptotic cascade in SW620 cells that was induced independently of growth arrest, dissipation of the deltapsim, ROS production, or synthesis of de novo RNA or protein, and we have linked its efficient initiation to early elevation of the deltapsim.

Initiation of growth arrest and apoptosis of MCF-7 mammary carcinoma cells by tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate, is associated with mitochondrial activity.

We investigated the effects of tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate and an approved food additive, establishing induction of growth arrest and apoptosis of MCF-7 human mammary carcinoma cells. Transient increased mitochondria-associated bax, dissipation of the mitochondrial membrane potential (delta(psi)m), and caspase-3-independent cleavage of poly(ADP-ribose) polymerase are evident as early as 4 h after treatment of cells with tributyrin. These events are followed by the transient accumulation of mitochondrial cytochrome c in the cytosol and, finally, the generation and accumulation of cells with subdiploid DNA content. During the period in which mitochondria-associated bax levels are elevated, the delta(psi)m is disrupted, and cytochrome c is detected in the cytosol, we show induction of p21WAF1/Cip1 in the absence of increased p53 and arrest of cells in G2-M. Thus, early mitochondria-associated events may play a key role in initiating and/or coordinating tributyrin-mediated growth arrest and apoptosis of wild-type p53 MCF-7 cells. Because effective chemoprevention has been associated with agents that restore or maintain the balance between proliferation and apoptosis, dietary tributyrin, particularly during the critical period of mammary gland development, may be a promising chemopreventive agent.

Mitochondrial membrane potential (delta psi(mt)) in the coordination of p53-independent proliferation and apoptosis pathways in human colonic carcinoma cells.

We have previously defined depressed mitochondrial function as a determinant in colon cancer risk and progression and established that metabolism of butyrate, a short-chain fatty acid generated during the fermentation of fiber by endogenous intestinal bacteria, induces mitochondrial function-dependent growth arrest and apoptosis of colonic carcinoma cells in vitro. Here, we dissect the relationships among mitochondrial function, growth arrest, and apoptosis, reporting that initiation and maintenance of butyrate-mediated p53-independent p21WAF1/Cip1 induction and subsequent G0/G1 arrest require an intact mitochondrial membrane potential (delta psi(mt)) and that the process of dissipation of the delta psi(mt) is then essential for initiation of a butyrate-induced apoptotic cascade. Thus, we hypothesize that mitochondria play a pivotal role in coordinating proliferation and apoptosis pathways, a coordination that must be tightly regulated in rapidly renewing tissues, such as the colonic mucosa.

Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function.

Butyrate, a short-chain fatty acid produced during microbial fermentation of fiber, induces growth arrest, differentiation, and apoptosis of colonic epithelial cells in vitro, and our prior work has shown that this induction is tightly linked to mitochondrial activity. Here we demonstrate that 12 h following induction, SW620 human colonic carcinoma cells accumulate simultaneously in G0-G1 and G2-M of the cell cycle. Four h later, during this G0-G1 to G2-M arrest, cells begin to undergo apoptosis. Using a series of unrelated agents that modulate mitochondrial functions, we demonstrate that mitochondrial electron transport and membrane potential are critical in initiation of this butyrate-mediated growth arrest and apoptosis. Colonic tumorigenesis is characterized by abnormalities in proliferation, apoptosis, and mitochondrial activities. Thus, butyrate may reduce risk for colon cancer by inducing a pathway that enhances mitochondrial function, ultimately resulting in initiation of growth arrest and apoptosis of colonic epithelial cells.

Steady-state levels of mitochondrial messenger RNA species characterize a predominant pathway culminating in apoptosis and shedding of HT29 human colonic carcinoma cells.

A differentiated human colonic epithelial cell has undergone relatively stable molecular, biochemical, and cellular alterations resulting in the acquisition of structures, activities, and functions that characterize it as one of at least three mature phenotypes: a columnar absorptive, secretory, or enteroendocrine cell. We have shown previously that induction of HT29 cells with the short-chain fatty acid sodium butyrate elevates alkaline phosphatase activity, a marker of the absorptive cell phenotype, and increases mitochondrial gene expression. Furthermore, this induction is accompanied by subsequent apoptosis and cell shedding. In this report, we have investigated the effects of forskolin, a potent inducer of the MUC2 gene in HT29 cells, a marker of the secretory phenotype, and have shown that neither apoptosis nor mitochondrial gene expression are significantly stimulated. Thus, differentiation along the secretory cell lineage may not play a major role in apoptosis of colonic epithelial cells. Moreover, we have also investigated two polar solvents, DMSO and dimethylformamide, which have been reported to induce a more differentiated, but as yet not well characterized, phenotype in colonic carcinoma cells in culture. Although neither polar solvent induces alkaline phosphatase expression or MUC2 expression, both induce significant apoptosis, again associated with significant elevation of mitochondrial gene expression. Thus, elevation of mitochondrial gene expression appears to be an important pathway in the induction of apoptosis in colonic epithelial cells in culture, whether or not markers characteristic of differentiation along either the absorptive or secretory cell lineage are induced.

Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines.

The architecture of normal colonic mucosa suggest that terminally differentiated epithelial cells near the top of the crypt are extruded into the colonic lumen. Morphological studies have identified apoptotic cells among the differentiated phenotypes near the crypt-lumen interface, suggesting a link between pathways of differentiation, apoptosis, and cellular shedding. We studied these processes in HT29 and SW620 cells and found that compared to adherent cells, those cells which were shed during standard, uninduced culture conditions exhibited nonrandom DNA fragmentation characteristic of apoptosis. Moreover, these apoptotic cells, which accumulate in the media, exhibited a more differentiated phenotype. Because short-chain fatty acids (SCFAs) are natural effectors of colonic cell differentiation in vivo, we investigated the specificity of three 4-carbon atom SCFAs on potentiating differentiation and apoptosis, and thus accumulation of shed cells in the conditioned media, in these colonic carcinoma cell lines. Whereas the unbranched SCFA butyrate induced a more differentiated phenotype and enhanced apoptosis, two derivatives of butyrate, branched isobutyric acid and a nonmetabolizable fluorine-substituted analogue, heptafluorobutyric acid, were ineffective in inducing either differentiation or apoptosis. Thus, potentiated differentiation and apoptosis in colonic carcinoma cells were linked to SCFA structure and, most likely, utilization.