The permeability transition pore (PTP) is a mitochondrial channel whose opening causes the mitochondrial membrane potential (ΔΨ) collapse that leads to apoptosis. Some ubiquinone analogues have been demonstrated previously to modulate the PTP open-closed transition in isolated mitochondria and thought to act through a common PTP-binding site rather than through oxidation-reduction reactions. We have demonstrated recently both in vitro and in vivo that the ubiquitous free radical scavenger and respiratory chain coenzyme Q₁₀ (CoQ₁₀) prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. On this basis, we hypothesized that the antiapoptotic property of CoQ₁₀ could be independent of its free radical scavenging ability and related to direct inhibition of PTP opening. In this study, we have verified this hypothesis by evaluating the antiapoptotic effects of CoQ₁₀ in response to apoptotic stimuli, serum starvation, antimycin A, and ceramide, which do not generate free radicals, in comparison to control, free radical-generating UVC irradiation. As hypothesized, CoQ₁₀ dramatically reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli. This was accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and caspase 9 activation. Because these events are consequent to mitochondrial PTP opening, we suggest that the antiapoptotic activity of CoQ₁₀ could be related to its ability to prevent this phenomenon.

Purpose: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB₁ and CB₂, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation.

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E₂ and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 μmol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer. [Mol Cancer Ther 2006;5(11):2716–26]

Recently, carbonic anhydrase (CA) inhibitors have been proposed as a potential new class of antitumor agents. The aim of this study was to evaluate the antitumor activity of three CA inhibitors, namely acetazolamide (AZ) and two newly synthesized aromatic sulfonamides with high affinity for CA IX, 2-(4-sulfamoylphenyl-amino)-4,6-dichloro-1,3,5-triazine (TR1) and 4-[3-(N,N-dimethylaminopropyl)thioreidophenylsulfonylaminoethyl]benzenesulfonamide (GA15), against human tumor cells. The effects of AZ, TR1, and GA15 on cell proliferation and apoptosis were evaluated in CA IX-positive HeLa and 786-O cells and CA IX-negative 786-O/von Hippel-Lindau (VHL) cells. We also investigated whether the potential antitumor activity of these molecules might be mediated by an increase in ceramide production. AZ, TR1, and GA15 could significantly reduce cell proliferation and induce apoptosis in HeLa and 786-O cells. Moreover, all three inhibitors could decrease intracellular pH (pH_i) and increase ceramide production in the same cells. Treatment with the ceramide synthase inhibitor fumonisin B1 prevented the apoptotic effects of the three CA inhibitors. In all experiments, the effects of aromatic sulfonamides were more pronounced than those of AZ. The three inhibitors did not show any antitumor activity in CA IX-negative 786-O/VHL cells and failed to lower pH_i or increase intracellular ceramide levels in the same cells. In conclusion, CA inhibition can decrease cell proliferation and induce apoptosis in human tumor cells. The ability of CA inhibitors to decrease pH_i might trigger cell apoptosis through mediation of ceramide synthesis. Activation of this apoptotic cascade probably is mediated by inhibition of the CA IX isoform.

We previously identified a conserved A + U-rich element (ARE) in the 3′-untranslated region of bcl-2mRNA. We have also recently demonstrated that the bcl-2ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2mRNA half-life reduction. Here we show that the AUBP AUF1 bindsin vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1–257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to thebcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2–3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.