Retinoid-related molecules induce cytochrome c release and apoptosis through activation of c-Jun NH(2)-terminal kinase/p38 mitogen-activated protein kinases.

Retinoid-related molecules have been described that induce apoptosis in a variety of cancer cell lines. Of particular interest is the apoptotic activity of the all-trans-retinoic acid receptor gamma-selective molecules MX2870-1 and MX3350-1. These compounds have been shown to be effective in vivo against lung cancer and could therefore serve as important leads for novel anticancer drugs. We analyzed the death signaling pathways activated by these molecules. We observed that apoptotic retinoid-related molecules (RRMs) cause the release of cytochrome c from the mitochondria and subsequent activation of caspases 9 and 3. This was preceded by a strong and sustained activation of c-Jun NH(2)-terminal kinase as well as p38 kinase, which was independent of caspase activity. Inhibition of p38 kinase activity by the specific inhibitor SB203580 did not affect the induction of apoptosis by MX2870-1. However, interference with the activation of c-Jun NH(2)-terminal kinase and p38 stress kinases by PD169316 completely blocked all signs of apoptosis, including caspase activity, DNA fragmentation, and phosphatidylserine externalization. PD169316 also prevented the cleavage of Bid and the release of cytochrome c induced by this class of RRMs. Furthermore, processing and activation of different caspases by MX2870-1 was completely inhibited by increasing concentrations of PD169316. Thus, the investigated RRMs induce a death pathway, which is independent of Fas ligand, that is also activated by UV radiation and other agents. Our findings open the possibility for the future use of this class of RRMs in combination therapies with other anticancer drugs.

Retinoid-related molecules require caspase 9 for the effective release of Smac and the rapid induction of apoptosis.

Certain retinoid-related molecules (RRMs) with agonist or antagonist activities have been described to induce apoptosis in a variety of cancer cell lines and show promise for the treatment of cancer. Similar to other chemotherapeutic drugs, these retinoid analogs have been suggested to induce apoptosis through the intrinsic pathway, which requires the release of cytochrome c from the mitochondria for the effective activation of caspase 9. Expression of a catalytically inactive form of caspase 9, which functions as a dominant negative mutant, inhibits the induction of DEVDase activity and nuclear fragmentation by selective RRMs. Whereas the RRMs could induce the release of cytochrome c in the absence of caspase 9 activity, the later is necessary for the effective release of Smac/Diablo from the mitochondria. Furthermore, overexpression of Bcl-2 or Bcl-X(L) also inhibits RRM-induced apoptosis. We demonstrate that activation of caspase 2 by the agonist MX2870-1 requires caspase 9 activity and is inhibited by Bcl-2 overexpression. In contrast, the antagonist MX781 induces cleavage of procaspase 2 upstream of mitochondria and independently of caspase 9. Thus, two retinoid analogs with unique characteristics activate two distinct apical caspases (2 or 9) to initiate apoptosis. In addition to caspase-mediated cell death, sustained exposure to the RRMs can also lead to loss of cell viability in cells lacking caspase 9 activity or in cells stimulated in the presence of the caspase inhibitor Z-VAD-fmk. Moreover, MX2870-1 and MX781 produce cell cycle arrest independently of caspase activity and the retinoid receptors.

A non-linear least-squares approach to the resolution of heterogeneous fluorescence from multitryptophan proteins.

Protein heterogeneous fluorescence results from the different microenvironment of each emitting chromophore. The structural and dynamic information contained in this emission can be extracted to some extent by selective quenching experiments. In this work, graphical and numerical methods are described for the analysis of protein emission in terms of three separated contributions: a fluorescence fraction which is not accessible to the quencher and two additional fractions with different solvent exposure. 'Static quenching' deviations from Stern-Volmer behaviour are also discussed. The application of these methods is exemplified on simulated quenching experiments and real data on acrylamide quenching of lysozyme fluorescence.

Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies.

Apoptosis is a mode of cell death through which cells are dismantled and cell remains are packed into small, membrane-bound, sealed vesicles called apoptotic bodies, which are easy to erase by phagocytosis by neighbouring and immune system cells. The end point of the process is to cleanly eliminate damaged or unnecessary cells without disrupting the surrounding tissue or eliciting an inflammatory response. The apoptotic process involves a series of specific events including deoxyribonucleic acid and nuclear fragmentation, protease-driven cleavage of specific substrates, which inhibits key survival functions and reorganizes the cell's structure, externalization of molecules involved in phagocytosis, membrane blebbing and cell shrinkage. Apoptotic volume decrease (AVD) leading to cell shrinkage is a core event in the course of apoptosis, the biological meaning of which has not been clearly ascertained. In this article we argue that volume loss is a geometrical requisite for cell dismantling into apoptotic bodies. This is derived from the cell's volume-to-surface ratio. Indeed, package of the original cell volume into smaller membrane-sealed vesicles requires that either cell membrane surface increase or cell volume decrease. In this sense, AVD provides a reservoir of membrane surface for apoptotic body formation. The strategic situation of AVD in the time course of apoptosis is also discussed in the context of apoptotic body formation.

Mesenteric cyclooxygenase products after combined antihypertensive treatment in uninephrectomized SHRs.

Unilateral nephrectomy in the spontaneously hypertensive rat (SHR) does not produce any change in blood pressure but does induce humoral alterations that might influence the antihypertensive action of some drugs. In this study, the antihypertensive effect of treatment (5 weeks) with placebo (control), verapamil, trandolapril, or their combination (verapamil plus trandolapril) was investigated in SHRs with half renal mass ablation, regarding the structure and function of small mesenteric arteries. Arterial pressure was followed during the period of treatment. Trandolapril and veratran returned pressure to normal, while verapamil was ineffective. Statistically significant differences in the parameters of vessel structure were not observed among groups; thus, the alterations in functionality cannot be attributed to morphologic changes. The noradrenaline-induced contraction was reduced similarly by the three treatments as compared to controls. This difference involved a higher participation of nitric oxide in the trandolapril group, while in the verapamil group the origin of the difference might be due to the abolishment of a cyclooxygenase product. Veratran retained both effects. Acetylcholine-evoked relaxation of vessels precontracted with noradrenaline was improved with treatments versus controls. The abolishment of a contracting prostanoid or an NO scavenger from the cyclooxygenase pathway, due to the treatments implemented, is probably the cause of this.

Antihypertensive action of trandolapril and verapamil in spontaneously hypertensive rats after unilateral nephrectomy.

It is well documented that the kidney plays a fundamental role in long-term arterial pressure regulation, and, as an endocrine organ, in the regulation of cardiovascular structure and functionality. In this study, the antihypertensive effect of long-term treatment (6 months) with placebo, verapamil, trandolapril, and a combination of the latter (verapamil plus trandolapril) was investigated in spontaneously hypertensive rats after half-renal-mass ablation. Arterial pressure was monitored during treatment and at the end, aortic structure and functionality were investigated. Trandolapril and the combination returned pressure to normal, whereas verapamil was less effective. All three treatment groups were similarly effective at reducing aortic medial hypertrophy, the wall-to-lumen ratio, and contraction evoked by potassium chloride and noradrenaline. Verapamil and veratran were more effective than trandolapril at reducing lamina media cross-sectional area. Trandolapril and the combination were more effective than verapamil at improving endothelial dysfunction.

Antihypertensive effect of trandolapril and verapamil in rats with induced hypertension.

The antihypertensive effect of long-term treatment (6 months) with placebo (as control), verapamil, trandolapril, and their combination (verapamil plus trandolapril) was investigated in Wistar rats rendered hypertensive by extensive renal mass ablation, as a model lacking genetic hypertensive determinants. Arterial pressure was monitored during treatment and at the end, aortic structure and functionality were investigated. Trandolapril and the combination prevented the increase in pressure observed in the control group after renal handicap, whereas verapamil was much less effective. Trandolapril and the combination were similarly effective, whereas verapamil was ineffective, or even deleterious, at reducing aortic lamina media hypertrophy, the wall-to-lumen ratio, lamina media cross-sectional area, potassium chloride-induced contraction, and at increasing acetylcholine relaxation. The response to noradrenaline decreased in the trandolapril group, increased in the verapamil group, and remained unmodified in the association group. In conclusion, treatment with trandolapril exerts beneficial antihypertensive actions in this model of induced hypertension, showing continuous control of blood pressure and prevention of structural and functional alteration of the aorta. Verapamil exerts weak control of arterial pressure and produces, if any, deleterious effects on the structure and function of the aorta. These negative effects of verapamil are overcome by coadministration of trandolapril.