Receptor activation and inhibition in cellular response to chemotherapeutic combinational mimicries: the concept of divergent targeting.

The antiproliferative effect of tandem somatostatin receptor (SSTR) activation, epidermal growth factor receptor (EGFR) inhibition, and induction of DNA damage was analyzed using octreotide (OCT), a SSTR agonist, the clinical DNA methylating agent temozolomide (TMZ), Iressa, an EGFR inhibitor, and dual EGFR-DNA targeting agents termed "combi-molecules". Using SSTR-expressing glioma cells harbouring low levels of EGFR (U87MG) or transfected to overexpress EGFR (U87/EGFR) or a variant (U87/EGFRvIII), we showed that Iressa, alone or in combination with the DNA damaging agent TMZ, and combi-molecules RA2 and RA5 inhibited EGF-induced phosphorylation of EGFR in U87MG and more moderately in U87/EGFR and U87/EGFRvIII transfected cells. This translated into equivalent levels of Erk 1/2 inhibition. Activation of SSTRs with OCT did not modulate the effects of the various treatments on Erk 1/2 phosphorylation. Likewise, SSTR activation did not alter TMZ- or DNA-damaging combi-molecules, RA2 and RA5, induced p53 activation nor upregulation. However, SSTR activation significantly shifted TMZ-, RA2- and RA5-induced cell-cycle arrest to earlier phases (i.e., G2/M to late S, late S to S, S to G1). Further analysis showed that apoptosis was not induced. This was in agreement with the fact that p53 activation did not induce Bax upregulation nor did EGFR inhibition promote Bad dephosphorylation. Moreover, enhancement of survivin, an anti-apoptotic protein, expression was observed. The results in toto suggest that the combination of SSTR activation with EGFR inhibition and DNA damage affects cell-cycle progression but a disconnection between the targeted signalling pathways in these brain tumour cells precludes synergistic cell-killing by the triple growth inhibitory events.

Somatostatin receptors in primary human breast cancer: quantitative analysis of mRNA for subtypes 1--5 and correlation with receptor protein expression and tumor pathology.

Somatostatin receptors (SSTRs) have been identified in most hormone-producing tumors as well as in breast cancer. In the present study, we determined SSTR1-5 expression in primary ductal NOS breast tumors through semi-quantitative RT-PCR and immunocytochemistry. The results from the analysis of 98 samples were correlated with several key histological markers and receptor expression. All five SSTR subtypes are variably expressed at the mRNA level in breast tumors with 91% of samples showing SSTR1, 98% SSTR2, 96% SSTR3, 76% SSTR4, and 54% SSTR5. SSTR1-5 are localized to both tumor cells and the surrounding peritumoral regions as detected by immunocytochemistry. Levels of SSTR mRNA, when corrected for beta-actin levels, were highest for SSTR3 followed by SSTR1, SSTR2, SSTR5, and SSTR4. Furthermore, there was good correlation between mRNA and protein expression with 84% for SSTR1, 79% for SSTR2, 89% for SSTR3, 68% for SSTR4, 68% for SSTR5, and 78% for all five receptors. SSTR1, 2 and 4 were correlated with ER levels whereas SSTR2 showed an additional correlation with PR levels. These correlations were independent of patient age and histological grade. Moreover, using immunocytochemistry, blood vessels exhibited receptor-specific localization for SSTR2 and SSTR5. Our results indicate significant correlations between mRNA and protein expression along with receptor-specific correlations with histological markers as well as ER and PR levels. Differential distribution of SSTR subtypes in tumors and receptor-specific expression in vascular structures may be considered as a novel diagnosis for breast tumors with receptor subtype agonists.

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1.

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH2-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST[1-10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys13 followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST[1-10] production. Since the prosomatostatin sequence R8-Q9-F10-L11 \ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST[1-10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST[1-10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST[1-10] required the RXRXXL motif. However, this NH2-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.