A peptide of SPARC interferes with the interaction between caspase8 and Bcl2 to resensitize chemoresistant tumors and enhance their regression in vivo.

SPARC, a matricellular protein with tumor suppressor properties in certain human cancers, was initially identified in a genome-wide analysis of differentially expressed genes in chemotherapy resistance. Its exciting new role as a potential chemosensitizer arises from its ability to augment the apoptotic cascade, although the exact mechanisms are unclear. This study further examines the mechanism by which SPARC may be promoting apoptosis and identifies a smaller peptide analogue with greater chemosensitizing and tumor-regressing properties than the native protein. We examined the possibility that the apoptosis-enhancing activity of SPARC could reside within one of its three biological domains (N-terminus (NT), the follistatin-like (FS), or extracellular (EC) domains), and identified the N-terminus as the region with its chemosensitizing properties. These results were not only confirmed by studies utilizing stable cell lines overexpressing the different domains of SPARC, but as well, with a synthetic 51-aa peptide spanning the NT-domain. It revealed that the NT-domain induced a significantly greater reduction in cell viability than SPARC, and that it enhanced the apoptotic cascade via its activation of caspase 8. Moreover, in chemotherapy resistant human colon, breast and pancreatic cancer cells, its chemosensitizing properties also depended on its ability to dissociate Bcl2 from caspase 8. These observations translated to clinically significant findings in that, in-vivo, mouse tumor xenografts overexpressing the NT-domain of SPARC had significantly greater sensitivity to chemotherapy and tumor regression, even when compared to the highly-sensitive SPARC-overexpressing tumors. Our results identified an interplay between the NT-domain, Bcl2 and caspase 8 that helps augment apoptosis and as a consequence, a tumor's response to therapy. This NT-domain of SPARC and its 51-aa peptide are highly efficacious in modulating and enhancing apoptosis, thereby conferring greater chemosensitivity to resistant tumors. Our findings provide additional insight into mechanisms involved in chemotherapy resistance and a potential novel therapeutic that specifically targets this devastating phenomenon.

AMP-activated protein kinase influences metabolic remodeling in H9c2 cells hypertrophied by arginine vasopressin.

Substrate use switches from fatty acids toward glucose in pressure overload-induced cardiac hypertrophy with an acceleration of glycolysis being characteristic. The activation of AMP-activated protein kinase (AMPK) observed in hypertrophied hearts provides one potential mechanism for the acceleration of glycolysis. Here, we directly tested the hypothesis that AMPK causes the acceleration of glycolysis in hypertrophied heart muscle cells. The H9c2 cell line, derived from the embryonic rat heart, was treated with arginine vasopressin (AVP; 1 microM) to induce a cellular model of hypertrophy. Rates of glycolysis and oxidation of glucose and palmitate were measured in nonhypertrophied and hypertrophied H9c2 cells, and the effects of inhibition of AMPK were determined. AMPK activity was inhibited by 6-[4-(2-piperidin-1- yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo-[1,5-a]pyrimidine (compound C) or by adenovirus-mediated transfer of dominant negative AMPK. Compared with nonhypertrophied cells, glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation in hypertrophied cells, a metabolic profile similar to that of intact hypertrophied hearts. Inhibition of AMPK resulted in the partial reduction of glycolysis in AVP-treated hypertrophied H9c2 cells. Acute exposure of H9c2 cells to AVP also activated AMPK and accelerated glycolysis. These elevated rates of glycolysis were not altered by AMPK inhibition but were blocked by agents that interfere with Ca(2+) signaling, including extracellular EGTA, dantrolene, and 2-aminoethoxydiphenyl borate. We conclude that the acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK, whereas the acute glycolytic effects of AVP are AMPK independent and at least partially Ca(2+) dependent.

Effects of E4031 and quinidine on atrial and ventricular refractoriness in rabbits in vivo.

This study assessed the effects of E4031 and quinidine on refractoriness (ERP) in a new in vivo model in rabbits. Following sinoatrial (SAN) and atrioventricular node (AVN) ablation ERP was determined in atria and ventricles with the shortest S1-S2 interval eliciting a second electrogram defined as the ERP. The effects of E4031 and quinidine (dose ranges 1-8 micromol/kg) were compared. E4031 dose-dependently increased ERP. The maximum change from pre-drug values with E4031 was 27+/-8 msec (a 36+/-12% increase) at 2 Hz in atria and 51+/-9 msec (27+/-5%) at 2 Hz in ventricles. Negative frequency-dependence was observed only in ventricles. Quinidine dose-dependently increased ERP. The maximum increase for quinidine was 23+/-3 msec (28+/-4%) at 2 Hz in atria and 25+/-10 msec (22+/-10%) at 6 Hz in ventricles, but without frequency-dependence in either tissue. In comparison to E4031, quinidine produced smaller changes in ERP and showed minimal frequency dependence. Thus, the added presence of sodium blocking actions with quinidine did not produce greater effects on ERP than I(Kr) blockade alone with E4031. However, quinidine also blocks other potassium currents, such as Ito, and the degree of I(Kr) blockade with E4031 was probably greater than that with the same dose of quinidine. This model may have clinical utility for testing multi-ion channel blocking drugs.

Effects of flecainide on atrial and ventricular refractoriness in rabbits in vitro and in vivo.

This study compared the in vitro versus in vivo effects of flecainide on effective refractory period (ERP) in atrial and ventricular tissue in rabbits. Flecainide (a class 1c agent) was chosen, on the basis of its known pharmacological profile and antiarrhythmic actions, to provide a reference compound for investigating models that suitably predict the clinical effects of antiarrhythmics. The rabbit models used were those previously described by Lowe et al. (2002) and Leung et al. (2003). ERP was measured as the shortest S1-S2 interval that elicited a second contraction (in vitro) or electrogram (in vivo). Flecainide (1-10 microM) in vitro produced a concentration-dependent increase in ERP. The greatest drug-induced change from pre-drug values in vitro occurred with the highest concentration in atria and ventricles at 4 Hz. The change was 30+/-4 msec (33+/-7%) in atria versus 53+/-8 msec (46+/-10%) in ventricles. In vivo, flecainide (1 - 4 micromol/kg) dose-dependently increased atrial ERP at 2 and 6 Hz. The biggest change was 28+/-17 msec (29+/-16%). However, there was no effect at 4 Hz. In the ventricles, a dose-related increase in ERP was only seen at 4 Hz (26+/-6 msec). Flecainide showed no frequency dependence of action on ERP in any preparation. Flecainide produced adverse effects both in vitro and in vivo. A concentration and frequency-dependent negative inotropic effect was seen in vitro, and dose-related hypotension in vivo. The highest dose (8 micromol/kg i.v.) of flecainide was lethal. Flecainide produced the expected electrophysiological and toxicity profile, both in vitro and in vivo. Despite such findings, the drug is used to terminate and prevent atrial arrhythmias clinically. In conclusion our rabbit models for determining ERP may not be useful in predicting the clinical usefulness of a drug like flecainide.