FaDu SN-38 concentration was significantly (p<0.05) increased and the percentage increase was 124%, when compared with irinotecan alone ( table 3). Irinotecan concentration was significantly (p<0.05) decreased and the percentage change was −60%, when compared with irinotecan alone ( table 3). In order to determine the effect of the second course treatment on intra-tumor SN-38 concentration, we studied the sensitive FaDu tumors. Panel C1 demonstrates that the second irinotecan treatment on day 14 also induces apoptotic tumor cells labeled by arrows and the combination treatment with MSC (panel C2) significantly increased the apoptosis incidence, as seen on the corresponding bar graphs. Panel B1 visualizes that the first CPT treatment on day 7 induced several apoptotic tumor cells indicated by arrows, but the combination treatment with MSC (panel B2) did not increase the apoptotic incidence significantly. Panel A1 illustrates that MSC did not change the incidence of apoptotic tumor cells, as compared to the control (panel A2). slides in randomly selected, non-necrotic fields (×400) of tumor and expressed as a percentage. Apoptotic tumor cell nuclei were counted among 300–400 tumor cell nuclei on H.E. Apoptotic cells were identified by morphology based on the characteristic nuclear fragmentation and condensation. The pictures are representative microphotographs of conventional, formalin-paraffin sections of the tumors after hematoxylin-eosin staining.
Double immunohistochemical staining for endothelial cells and pericytes in tumors Quality assurance was maintained by simultaneously assaying the quality control samples prepared in bulk, prior to assay validation. The limit of quantitation for both was 2.5ng/ml. Detection was by fluorescence, with excitation at 370nm and emission at 510nm. The separation method was carried out on a Waters Nova-Pak C18 column equipped with a ♛ondapak C18 guard column, with the mobile phase consisting of 20% acetonitrile and 80% triethylamine acetate. The lactone forms of irinotecan and its active metabolite, SN-38, were measured using a validated HPLC method with fluorescence detection as described by Warner and Burke. Protein measurement was performed on the pellet using Bradford protein assay. After centrifugation of the homogenate, the supernatant was evaporated to dry, reconstituted to mobile phase and subjected to HPLC. Samples were homogenized using a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY). Tissue samples were immediately incubated with ice-cold 5ml methanol and acetonitrile (1:1). Plasma was obtained at 4☌ immediately following the blood collection and subjected to the same solvent extraction procedure and HPLC. Tumors, blood and normal tissue samples were collected 2h after irinotecan alone or in combination.
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